Methods for treatment of and prophylaxis against inflammatory disorders

ABSTRACT

An isolated and purified peptide, neonatal NET-inhibitory Factor (nNIF), is disclosed. Methods for treatment of and prophylaxis against inflammatory disorders are also disclosed, including methods of treatment of and prophylaxis against inflammatory disorders comprising administering NET-inhibitory peptides (NIPs), which may be a nNIF, a pharmaceutically acceptable salt of a nNIF, a nNIF analog, a pharmaceutically acceptable salt of a nNIF analog, a nNIF-Related Peptide (nNRP), including the nNRP, Cancer-Associated SCM-Recognition, Immune Defense Suppression, and Serine Protease Protection Peptide (CRISPP), a pharmaceutically acceptable salt of a nNRP, a nNRP analog, or a pharmaceutically acceptable salt of a nNRP analog, to an individual.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/441,982 filed Feb. 24, 2017, which is a divisional of U.S. patentapplication Ser. No. 14/904,048 filed Jan. 8, 2016, which is a NationalStage of International Application No. PCT/US2014/45597 filed Jul. 7,2014, and which claims priority to U.S. Provisional Application No.61/843,618 filed Jul. 8, 2013, each of which are herein incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Numbers K08HD049699, R01 HL044525 and R01 HL066277 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Jun. 19, 2019 as a text file named“21101_0371U4_Updated_Sequence_Listing.txt,” created on Apr. 3, 2019,and having a size of 2,497 bytes is hereby incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

TECHNICAL FIELD

The present disclosure is directed to methods for treatment of andprophylaxis against inflammatory disorders.

BACKGROUND

Neutrophil extracellular traps (NETs) are extracellular lattices ofdecondensed chromatin decorated with antimicrobial proteins extruded bypolymorphonuclear leukocytes (PMNs, neutrophils) to trap and killmicrobes. Although NETs aid in trapping bacteria and other pathogens,their presence also leads to inflammatory tissue damage. Indeed, NETformation contributes to the pathology of several inflammatory disordersincluding acute lung injury resulting from influenza or bloodtransfusions, sepsis, small vessel vasculitis, systemic inflammatoryresponse syndrome (SIRS), and chronic autoimmune diseases like systemiclupus erythematosus. Furthermore, NET formation and, in particular, therelease of NET-associated histones into the extracellular space directlyinduces both epithelial and endothelial cell death in culture.

An active cell death process distinct from necrosis and apoptosis,frequently termed “NETosis,” leads to formation of three dimensionallattices studded with granule enzymes and host defense peptides thatbind to nuclear chromatin before extrusion from the neutrophil.Histones, which have antimicrobial activities, are also abundant inNETs. NET formation is conserved in many species.

Deficient NET formation is a mechanism of immunodeficiency and impairedhuman host defense. A variety of pathogens induce NET formation. Inaddition to Escherichia coli, Staphylococcus, Streptococcus, Yersinia,and other gram positive and negative bacteria, fungi, parasites, andviruses trigger generation of NETs by human PMNs. Lipopolysaccharides(LPS) also induce NETosis, suggesting that microbial toxins may broadlyhave this activity. Endogenous host mediators, including interleukin-8(IL-8), platelet activating factor (PAF), and complement factor C5ainduce NET formation directly or after “priming” by other mediators. LPSstimulated mouse platelets, activated human platelets, and platelets ina murine model of transfusion-related acute lung injury (TRALI) thatinvolves LPS priming also trigger NET formation. H1N1-infected alveolarepithelial cells induce NET formation by murine neutrophils in vitro.Thus, multiple interactions between neutrophils and microbes, hostcells, and/or host mediators signal NETosis and NET formation.

NETs are also major biologic instruments of extravascular microbialcontainment and killing in vitro and in vivo, thus limiting the spreadof pathogens. Certain pathogens, however, express endonucleases thatcleave the DNA lattice or inhibitors that block antimicrobial peptides;these mechanisms act as virulence factors that limit killing and providemechanisms for bacterial escape.

Failed NET formation appears to be a previously-unrecognized innateimmune deficit that results in severe infections. Patients with chronicgranulomatous disease (CGD), who are deficient in reactive oxygenspecies (ROS) generation and acquire recurrent, often life threateningbacterial and fungal infections, also demonstrate a defect in NETformation. Gene therapy for this immune deficiency can restore NETformation and control refractory pulmonary aspergillosis in patientswith CGD. This suggests that NETs can also demonstrate protectivefunctions in cystic fibrosis and pneumonia.

While the intracellular signaling pathways that regulate NET formationby PMNs remain largely unknown, ROS generation is considered a keyevent. Studies in human HL-60 myeloid leukocytes and genetically-alteredmice indicate that activity of peptidylarginine deiminase 4 (PAD 4), anenzyme responsible for chromatin decondensation, is also required.

Additionally, NET formation may require enzymatic activity of neutrophilelastase (NE) to initiate degradation of core histones leading tochromatin decondensation prior to plasma membrane rupture. Alpha 1anti-trypsin (A1AT) is a serine protease inhibitor that inactivates NEin plasma; it is, however, not expressed by human PMNs. A related serineprotease inhibitor, serpin B1, which is expressed as a cytoplasmicprotein by human PMNs, has been shown to restrict NET formation by mouseand human PMNs. Furthermore, treatment with recombinant serpin B1inhibits NET formation in human PMNs stimulated with phorbol12-myristate acetate (PMA), a robust inducer of NET formation in vitro.Recombinant A1AT, however, does not inhibit NET formation. Inhibition ofspecific serine proteases such as NE may effectively inhibit NETformation by human PMNs.

Although NET formation is a critical innate antimicrobial function ofPMNs, there is now clear evidence that it is a mechanism of inflammatorytissue injury and thrombosis if inappropriately triggered and/ordysregulated. See Saffarzadeh and Preissner, Curr Opin Hematol, 2013,20: 3-9; and Brinkmann and Zychlinsky, J Cell Biol, 2012, 198(5):773-783. NETs mediate inflammatory damage in multiple models of sterileand infectious challenge. For example, NET formation may be a keymechanism in the systemic vasculopathy that is central to thepathogenesis of the acute, pro-inflammatory phase of sepsis. Bacteriaincluding Staphylococci and E. coli are major causes of severe sepsis,alone or as polymicrobial infections, depending on the populationsstudied.

Experiments utilizing human endothelial cells (EC) and neutrophils invitro, and an in vivo model of endotoxemia in which mice were challengedwith LPS, indicate that NETs cause endothelial and liver damage,potentially mediated by neutrophil proteases associated with NETs. NEand other granule enzymes from neutrophils can potently injureendothelium and many extravascular cell types. EC activation, as occursin sepsis, can enhance NET generation. In addition to granule enzymes,histones associated with NETs are previously-unrecognized agonists forendothelial injury in sepsis, based on experimental models and humansamples.

There is also evidence that NETs and NET components are potentprocoagulants, and that NET components induce thrombosis—a centralpathogenetic feature of sepsis. NET components modify fibrin stabilityand fibrinolysis. Thus, while formation of NETs may be critical forbacterial capture and containment in the early phases of bacteremia andsepsis based on murine models, observations to date indicate that NETformation also causes damage to the host (for example, in acute septicsyndromes).

Activated vascular endothelium may induce NET formation by human PMNsand lead to endothelial cell damage in vitro. Also, cellular damage mayoccur when human endothelial cells are incubated with activatedplatelets and PMNs, leading to NET formation, and liver injury may occurin vivo following NET formation. Finally, placentas from mothers withsevere pre-eclampsia, a syndrome of pregnancy commonly leading topremature infant delivery, show exuberant NET formation. Thus, whileessential in preventing severe infections, inappropriate NET formationappears to also be a mechanism of inflammatory vascular and tissueinjury.

Inflammatory and infectious pulmonary syndromes provide another exampleof NET-mediated tissue injury. NETs form and contribute to vascular andalveolar dysfunction in animal models of acute lung injury and adultrespiratory distress syndrome (ARDS). ARDS is a major complication ofhuman systemic and pulmonary infection and inflammation. NETs areassociated with acute lung injury in models of influenza-inducedpneumonitis, a common and lethal infectious trigger for ARDS. In vitrostudies indicate that NET histones may be critical mediators of alveolarendothelial and epithelial cell death.

In addition to sepsis and pulmonary injury, vasculitic syndromes are yetanother example in which NETs play pathogenetic roles. As in sepsis,NETs may mediate both vascular inflammation and thrombosis invasculitis. A variety of inflammatory stimuli and infectious agents cancause vasculitis.

Dysregulated inflammation has also been found to contribute to thepathogenesis of all the major complications of prematurity: necrotizingenterocolitis (NEC), respiratory distress syndrome (RDS), pneumonia,bronchopulmonary dysplasia (BPD), neonatal chronic lung disease (CLD),neurodevelopmental delay, retinopathy of prematurity (ROP), and sepsis.Neonatal CLD causes significant morbidity and mortality in the U.S. CLDis a complication of preterm birth that results from prolongedmechanical ventilation required for chronic respiratory failure. Itoccurs in up to 70% of mechanically ventilated extremely low birthweight infants (ELBW) with respiratory distress. While surfactanttherapy and pre- or postnatal steroids have decreased the severity ofCLD, this significant morbidity associated with preterm birth remainscommon in at-risk infants, with about 8,000 to 10,000 new casesoccurring annually in the U.S. The mortality rate due to CLD in ELBWinfants remains high from 15-60%. Furthermore, CLD remains the mostcommon cause of long-term hospitalization in neonates and is alsoassociated with developmental delay.

Additional current evidence strongly supports the conclusion that NETsare involved in tissue damage and thrombosis in a variety ofinflammatory syndromes. Consequently, a few therapeutic strategies toblunt or interrupt NET formation or the activities of NET componentshave been investigated. All such strategies identified to date havemajor limitations, however. Global inhibition of neutrophil functionand/or specific inhibitors of oxygen radical generation or key molecularcheckpoints such as HIF-1α depress other PMN functions, includingmigration, phagocytosis, and/or intracellular microbial killing, causingparallel potential for microbial evasion and iatrogenic infections.

Disruption of NETs with DNases, potentially together with inhibition ofNET-associated histones and enzymes as a combination strategy,represents a therapeutic approach based on experimental models.Nevertheless, more than twenty different NET-associated proteins havebeen identified, making this impractical. Furthermore, enzymaticdisruption of NETs as an intervention may lead to dissemination ofmicrobes, depending on its timing. Pharmacologic disruption of NETs inthe vasculature also has the potential to spread histones and toxicneutrophil enzymes to other vascular beds, initiating or amplifyingmultiple organ injury. Finally, heparins inhibit NET formation undersome conditions, but have bleeding as a well-known complication.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments disclosed herein will become more fully apparent fromthe following description and appended claims, taken in conjunction withthe accompanying drawings. These drawings depict only typicalembodiments, which will be described with additional specificity anddetail through use of the accompanying drawings in which:

FIG. 1A is an image of adult human PMNs extruding NETs (thin arrows) totrap and kill bacteria, S. aureus (wide arrows).

FIG. 1B compares the decoration of NETs (thin arrow) with neutrophilelastase (NE) (thick arrow) formed by adult human PMNs to neonatal PMNswhich do express NE but do not form NETs.

FIG. 1C is a graph indicating that neonatal PMNs demonstratesignificantly decreased total and NET-mediated bacterial killing ascompared to adult PMNs. * p<0.05, ** p<0.001.

FIG. 2A is an image of NET formation by human PMNs stimulated with LPS(1 hour time point).

FIG. 2B is an image of human PMNs incubated in control plasma (1 hourtime point).

FIG. 2C is an image of NET formation by human PMNs incubated in plasmaisolated from patients with S. aureus sepsis (1 hour time point).

FIG. 3A is a series of images showing LPS-stimulated preterm PMNsisolated from the same preterm infant. PMNs were isolated from cordblood, at day of life 3, and day of life 14, as indicated.

FIG. 3B is a series of images (left to right) showing the results ofpre-incubation experiments: day of life 28 PMNs pre-incubated inautologous plasma, day of life 28 PMNs pre-incubated with cord bloodplasma, adult PMNs pre-incubated with autologous plasma, and adult PMNspre-incubated with cord blood plasma. NET formation was then inducedwith LPS stimulation.

FIG. 3C is a graph depicting extracellular histone H₃ release.Extracellular histone content (fold change over baseline) is representedon the y-axis. Data from one preterm infant's PMNs through day of life28 and one adult's PMNs from the plasma switch experiments of FIG. 3Bare represented on the x-axis.

FIG. 4A is a list of nNIF candidate proteins, expected molecular mass,and protein score.

FIG. 4B is a western blot using a polyclonal antibody against thecarboxy-terminus of A1AT comparing nNIF (≈6 kD) expression in cord bloodand adult plasma.

FIG. 4C is a comparison of the mass spectroscopy obtained sequences ofnNIF, CRISPP, and the A1ATm³⁵⁸ cleavage fragment of full length A1AT.

FIG. 5A is an image of untreated control adult PMNs.

FIG. 5B is an image of adult PMNs with LPS treatment.

FIG. 5C is an image of adult PMNs with LPS treatment followingpre-incubation with untreated cord blood plasma.

FIG. 5D is an image of adult PMNs with LPS treatment followingpre-incubation with nNIF-depleted cord blood plasma.

FIG. 5E is an image of adult PMNs with LPS treatment followingpre-incubation with affinity purified nNIF from cord blood plasma.

FIG. 5F is an image of adult PMNs with LPS treatment followingpre-incubation with full length rA1AT.

FIG. 6A is a series of images (clockwise from top left) showing adulthuman PMN NET formation in: control PMNs, PMA-stimulated (20 nM) PMNs,PMA-stimulated (20 nM) PMNs with scrambled peptide (1 nM)pre-incubation, and PMA-stimulated (20 nM) PMNs with CRISPP (1 nM)pre-incubation.

FIG. 6B is a graph showing the results of a human histone H₃ releaseassay to quantify NET formation. Extracellular histone content (foldchange over baseline) is represented on the y-axis. Data from human PMNsstimulated under indicated conditions are represented on the x-axis.

FIG. 7A is a series of images (clockwise from top left) showing humanPMN NET formation by: no treatment, S. aureus incubation, S. aureusincubation and scrambled control peptide (1 nM) pre-incubation, and S.aureus incubation and CRISPP (1 nM) pre-incubation.

FIG. 7B is a graph showing the results of a human histone H₃ releaseassay to quantify NET formation (n=2). Extracellular histone content(fold change over baseline) is represented on the y-axis. Data fromhuman PMNs stimulated under indicated conditions are represented on thex-axis.

FIG. 8A is a series of images (left to right) showing NET formation inhuman PMNs incubated with: LPS (100 ng/mL), dengue virus media withoutinfection, and dengue virus (0.05 MOI).

FIG. 8B is a series of images showing (clockwise from top left) humanPMNs incubated with: dengue virus media without infection and pretreatedwith scrambled control peptide (1 nM), dengue virus media withoutinfection and pretreated with CRISPP (1 nM), dengue virus and pretreatedwith CRISPP (1 nM), and dengue virus and pretreated with scrambledcontrol peptide (1 nM).

FIG. 9 is a graph showing total, phagocytotic, and NET-mediatedextracellular bacterial killing of a pathogenic strain of E. coli byhuman PMNs with or without LPS stimulation (100 ng/mL). PMNs were alsopretreated with CRISPP (1 nM), or a scrambled peptide control (1 nM). *denotes statistical significance (p<0.05).

FIG. 10A is a graph tracking survival of C57BL6 mice in varioustreatment arms following intraperitoneal injection of LPS (20 mg/kg).CRISPP-treated mice (10 mcg/kg/dose) received 2 doses intraperitoneally;one given 1 hour prior to infection and one dose given 6 hours afterinfection. The same dose, delivery route, and schedule were followed forthe scrambled peptide control mice. Six mice were assessed in bothgroups. The LPS plus CRISPP group survival was statistically greaterthan the LPS plus scrambled peptide group (p=0.02).

FIG. 10B is a graph tracking survival of outbred Swiss mice in varioustreatment arms following intraperitoneal injection of E. coli (4×10⁷bacteria). As in FIG. 10A, CRISPP-treated mice (10 mcg/kg/dose) received2 doses intraperitoneally; one given 1 hour prior to infection and onedose given 6 hours after infection. The same dose, delivery route, andschedule were followed for the scrambled peptide control mice. Here, 10mice were assessed in each group: no treatment, E. coli plus CRISPP, andE. coli plus scrambled peptide. The E. coli plus CRISPP group survivalwas statistically greater than the E. coli plus scrambled peptide group(p<0.0001).

FIG. 11 is a graph tracking survival of outbred Swiss mice in varioustreatment arms of a murine CL/P model. CRISPP treated mice (10 mcg/kg)received 2 doses intraperitoneally; one given 1 hour prior to surgery orsham surgery and one dose given 6 hours after surgery. The same dose,delivery route, and schedule were followed for the scrambled peptidecontrol mice. Ten mice were assessed in each of the CL/P groups. TheCL/P plus CRISPP group survival approached statistical significancecompared with the CL/P—control group (p=0.06).

FIG. 12A is an image showing NET formation in paraffin-embedded humanlung tissue obtained at autopsy of neonates who died with CLD.Extracellular, alveolar histone H₃ accumulation consistent with NETformation is indicated by the white arrow.

FIG. 12B is a series of images assessing NET formation inparaffin-embedded preterm lamb lung tissue in a sheep model of CLD.Referring to the top two images, extracellular, alveolar NET formationis indicated by the white arrows. Referring to the bottom two images,H&E staining demonstrates other hallmarks of neonatal CLD includinghypercellularity and alveolar simplification.

FIG. 13A is a series of images showing PMNs isolated from preterm Iambcord blood and from mature ewes. Control samples, and samples stimulatedwith LPS (100 ng/mL) for 1 hour are shown, as indicated.

FIG. 13B is a series of images showing mature ewe PMNs pretreated withCRISPP (1 nM) or a scrambled control peptide (1 nM) for 1 hour prior toLPS-stimulation (100 ng/mL), as indicated.

FIG. 14A is a series of images showing LPS-stimulated preterm PMNsisolated from the same preterm infant. PMNs were isolated at day of life0, day of life 3, day of life 14, and day of life 28, as indicated.

FIG. 14B is a graph depicting extracellular histone H₃ release.Extracellular histone content (fold change over baseline) is representedon the y-axis. Data from 7 preterm infant's PMNs through day of life 28are represented on the x-axis. * denotes p<0.05 and ** denotes p<0.01compared to the control (dashed line), arbitrarily set at 1.

FIG. 14C is a graph depicting extracellular histone H₃ release.Extracellular histone content (fold change over baseline) is representedon the y-axis. Data from five preterm infant's PMNs on day of life 28and five adult's PMNs from the plasma switch experiments of FIG. 3B arerepresented on the x-axis. * denotes p<0.05 LPS/Adult versusLPS/Preterm, ** denotes p<0.01 LPS/Preterm versus cord bloodLPS/Preterm, and † denotes p<0.001 LPS/Adult versus cord bloodLPS/Adult.

FIG. 15A is a western blot using a polyclonal antibody against thecarboxy-terminus of alpha 1-antitrypsin (AAT, also referred to herein asA1AT) comparing nNIF (≈4-6 kD) expression in cord blood (CB) and adultplasma (A).

FIG. 15B is a graph quantitating the results of FIG. 15A. * denotesp<0.05.

FIG. 15C is a series of images (left to right) showing adult human PMNNET formation in: control PMNs, LPS-stimulated PMNs, AAT pre-incubatedLPS-stimulated PMNs, nNIF pre-incubated LPS-stimulated PMNs, andscrambled control peptide (SCR) pre-incubated LPS-stimulated PMNs.

FIG. 16A is a series of images (clockwise from top left) showing NETformation in: control PMNs, LPS-stimulated (100 ng/mL) PMNs, nNIF (1 nM)pre-incubated LPS-stimulated PMNs, CRISPP (1 nM) pre-incubatedLPS-stimulated PMNs, and SCR (1 nM) pre-incubated LPS-stimulated PMNs.

FIG. 16B is a graph depicting extracellular histone H₃ content (foldchange over baseline) on the y-axis. Data from human PMNs underindicated conditions are represented on the x-axis. The dashed linerepresents the control values, arbitrarily set at 1. ** denotes p<0.05for LPS and SCR/LPS compared to control, and t denotes p<0.05 forCRISPP/LPS and nNIF/LPS compared to both the LPS and SCR/LPS groups.

FIG. 16C is a graph depicting extracellular histone H₃ content (foldchange over baseline) on the y-axis. Data from human PMNs underindicated conditions are represented on the x-axis. The dashed linerepresents the control values, arbitrarily set at 1. * denotes p<0.05for CRISPP/PMA compared to PMA and SCR/PMA groups.

FIG. 16D is a graph showing the results of a human histone H₃ releaseassay to quantify NET formation (n=2). Extracellular histone content(fold change over baseline) is represented on the y-axis. Data fromhuman PMNs stimulated under indicated conditions are represented on thex-axis.

FIG. 17A is a graph depicting mononuclear cell counts in peritonealfluid under indicated conditions. * denotes p<0.05 for CTL versus allother groups.

FIG. 17B is a graph depicting neutrophil cell counts in peritoneal fluidunder indicated conditions. * denotes p<0.05 for CTL versus all othergroups and t denotes p<0.05 for CRISPP/E. coli versus SCR/E. coli and E.coli groups.

FIG. 17C is a graph depicting E. coli colony-forming units (cfu) inperitoneal fluid under indicated conditions. * denotes p≤0.05.

FIG. 17D are images depicting NET formation in peritoneal fluidfollowing E. coli injection±pre-injection of CRISPP (10 mg/kg) or SCR(10 mg/kg), as indicated.

FIG. 17E is a graph depicting histone H₃ release to assess NETformation. Extracellular histone content (fold change over baseline) isrepresented on the y-axis. The dashed line represents the controlvalues, arbitrarily set at 1. * denotes p<0.05 for CRISPP/E. coli andnNIF/E. coli groups compared to E. coli and SCR/E. coli groups.

FIG. 17F are images depicting NET formation on peritoneal tissuefollowing E. coli injection±pre-injection of CRISPP (10 mg/kg) or SCR(10 mg/kg), as indicated.

FIG. 17G is a graph depicting quantitation of NET formation using IMAGEJsoftware and a standardized grid in 5 randomly selected visual fieldsfor each sample on confocal microscopy. The graph represents the numberof NETs crossing the standardized grid lines shown on the y-axis and theexperimental groups shown on the x-axis. * denotes p<0.05 for LPS v.Control, ** denotes p<0.01 for SCR/LPS group v. Control, and † denotesp<0.05 for CRISPP/LPS and nNIF/LPS groups v. SCR/LPS.

FIG. 18A is a graph depicting chemotaxis induced by IL-8 (2 ng/mL) forPMNs isolated from healthy adults following treatment±nNIF, CRISPP, orSCR peptide control, as indicated. The ability of nNIF and CRISPP toinduce chemotaxis on their own was also measured (CRISPP-BLW, NIF-BLW,and SCR-BLW columns). The y-axis shows fold change over baseline of PMNchemotaxis±standard error of the mean (SEM).

FIG. 18B is a graph depicting phagocytosis by PMNs isolated from healthyadults following a four hour incubation with fluorescently labelled E.coli BIOPARTICLES (6×10⁶ particles/mL). The y-axis depicts the percentof PMNs positive for intracellular E. coli±SEM as assessed via confocalmicroscopy with multi-plane assessment. Cytochalasin B and D (10 μM)pretreatment was used as a control for phagocytosis inhibition. *denotes p<0.05.

FIG. 18C is a graph depicting respiratory burst activity inLPS-stimulated PMNs (100 ng/mL)±pre-incubation for 1 hour with CRISPP (1nM) or SCR (1 nM). ROS generation was measured using a dihydrorhodamineassay. The columns indicate ROS generation shown as percent gatedevents±SEM.

FIG. 18D is a graph depicting total, phagocytic, and extracellularNET-mediated bacterial killing by LPS-stimulated human PMNs (100ng/mL)±pretreatment with CRISPP (1 nM) or SCR (1 nM). * denotes p<0.05and ** denotes p<0.01.

FIG. 18E is a graph depicting platelet p-selectin expression followingpre-incubation with CRISPP (1 nM) or SCR (1 nM) prior to thrombinstimulation (0.1 IU/mL), as indicated. The dashed line indicates thelevel of platelet p-selectin expression at baseline. † denotes p<0.001for CRISPP and SCR groups compared with all three thrombin-stimulatedgroups and baseline.

FIG. 18F is a graph depicting LPS-stimulated PMN aggregation withthrombin (0.1 IU/mL) activated platelets±CRISPP (10 nM) or SCR (10 nM)pre-incubation. The dashed line represents the LPS control group. *denotes p<0.05 for CRISPP/Factor IIa and SCR/Factor IIa groups comparedto baseline.

FIG. 18G is a series of images depicting CRISPP-FLAG Tagged (1 nM) andSCR-FLAG Tagged (1 nM) uptake by PMNs, co-incubated platelets and PMNs,and platelets, as indicated.

FIG. 19A is a series of images depicting the nuclear area of PMNspre-incubated with CRISPP (1 nM), SCR (1 nM), or Sivelestat (200 nM)prior to stimulation with PMA (20 nM), as indicated. White arrowheadshighlight decondensed nuclei.

FIG. 19B is a graph quantifying the results of the assays depicted inFIG. 19A. ** denotes p<0.01 and *** denotes p<0.001.

FIG. 19C is a series of images showing FLAG-tagged CRISPP and SCRpeptide cellular localization, as indicated.

FIG. 19D is a series of images depicting neutrophil elastase andCRISPP-F peptide co-localization. The control image (Co) depicts PMNstreated with both antibodies but without FLAG-tagged peptide treatment.

FIG. 20 is a graph depicting an assessment of ten mice in each of fourgroups: no treatment, E. coli, CRISPP-Post/E. coli, and SCR-Post/E.coli.

FIG. 21A is a series of images showing NET formation followingLPS-stimulation±pretreatment with CRISPP, CRISPP-F, or SCR-F, asindicated.

FIG. 21B is a graph showing the results of a histone H₃ release assay toquantify NET formation in response to LPS stimulation followingpre-incubation with SCR, CRISPP, or CRISPP-FLAG. Extracellular histonecontent (fold change over baseline±SEM) is represented on the y-axis. *denotes p<0.05.

DETAILED DESCRIPTION

This disclosure is related to methods for treatment of and prophylaxisagainst inflammatory disorders. It will be readily understood that theembodiments, as generally described herein, are exemplary. The followingmore detailed description of various embodiments is not intended tolimit the scope of the present disclosure, but is merely representativeof various embodiments. Moreover, the order of the steps or actions ofthe methods disclosed herein may be changed by those skilled in the artwithout departing from the scope of the present disclosure. In otherwords, unless a specific order of steps or actions is required forproper operation of the embodiment, the order or use of specific stepsor actions may be modified.

Each and every patent, report, and other reference recited herein isincorporated by reference in its entirety.

DEFINITIONS

A “NET-Inhibitory Peptide (NIP)” is an anti-inflammatory agent thatinhibits neutrophil extracellular trap (NET) formation. Examples of NIPsinclude, but are not limited to: a neonatal NET-Inhibitory Factor(nNIF); a pharmaceutically acceptable salt of a nNIF; an analog of anaturally occurring form of nNIF, which nNIF analog inhibits NETosisand/or the formation of NETs and is structurally altered, relative to agiven human nNIF, by at least one amino acid addition, deletion,substitution, or by incorporation of one or more amino acids with ablocking group; a pharmaceutically acceptable salt of a nNIF analog; anNIF-Related Peptide (nNRP); a pharmaceutically acceptable salt of anNRP; a nNRP analog; or a pharmaceutically acceptable salt of a nNRPanalog.

A “neonatal Neutrophil Inhibitory Factor peptide” or “nNIF peptide” isdefined herein as a nNIF which is naturally occurring in mammals.

A “neonatal NIF-related peptide” or “nNRP” is defined herein as aCancer-Associated SCM-Recognition, Immune Defense Suppression, andSerine Protease Protection Peptide (CRISPP) which is naturally occurringin humans; A1ATm³⁵⁸, which has been shown to inhibit NET formation; andother nNIF-related peptides; and as analogs of naturally occurring formsof nNRPs that inhibit NETosis and/or the formation of NETs and arestructurally altered, relative to a given human nNRP, by at least oneamino acid addition, deletion, substitution, or by incorporation of oneor more amino acids with a blocking group.

“Inflammatory disorders” are defined herein as disorders characterizedby pathological inflammation. Inflammatory disorders include, but arenot limited to, conditions associated with infection, autoimmunity, andallergy. Inflammatory disorders as defined herein may include, but arenot limited to, acute respiratory distress syndrome (ARDS),bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease(COPD), cystic fibrosis, inflammation in cancer and its complications,inflammatory bowel disease (IBD), inflammatory lung disease (ILD),influenza-induced pneumonitis, necrotizing enterocolitis (NEC), neonatalchronic lung disease (CLD), periodontitis, pre-eclampsia, retinopathy ofprematurity (ROP), sepsis, systemic inflammatory response syndrome(SIRS), thrombosis, transfusion-related acute lung injury (TRALI),vasculitis, autoimmune syndromes including, but not limited to,rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), andWegener's granulomatosis (WG), and disorders of nonresolvedinflammation. There are other inflammatory disorders not listed hereinbut known to those skilled in the art. For example, see Kumar et al.,Robbins and Cotran Pathologic Basis of Disease, pp. 43-77, 8th Edition,2010, Saunders Elsevier, Philadelphia, Pa.; Nathan, Nature, 2002, 420:846-852; and Amulic et al., Annu Rev Immunol, 2012, 30: 459-489.

The phrase “does not globally depress polymorphonuclear leukocyte (PMN)function,” when used in connection with a NIP, means that although theNIP may inhibit or substantially inhibit NETosis, the NIP does notinhibit or substantially inhibit other PMN functions. Other PMNfunctions include, but are not limited to, chemotaxis, chemokinesynthesis and secretion, cytokine synthesis and secretion, extracellularbacterial killing, intracellular bacterial killing, phagocytosis, and/orreactive oxygen species (ROS) generation. Methods of assaying thesefunctions are known in the art. For example, Example 11 describesmethods of assaying phagocytic bacterial killing.

Methods

This disclosure relates to therapeutic and related uses of NETInhibitory Peptides (NIPs), neonatal NET-Inhibitory Factors (nNIFs),nNIF analogs, nNIF-Related Peptides (nNRPs), and nNRP analogs,particularly for inhibiting NETosis and/or the formation of neutrophilextracellular traps (NETs).

A first aspect of the disclosure relates to methods of treatinginflammatory disorders.

In certain embodiments, this disclosure provides methods of treating apatient having an inflammatory disorder comprising administering to thepatient an effective amount of a pharmaceutical composition comprising aNIP, or a pharmaceutically acceptable salt of a NIP, and apharmaceutically acceptable carrier to reduce a pathological effect orsymptom of the inflammatory disorder. The pathological effects orsymptoms may include one or more of the following: pain, heat, redness,swelling and/or edema, hypotension, fibrosis and/or post-inflammatoryfibrosis, end organ failure (i.e., renal, cardiac, hepatic), tissuedamage, and/or loss of function.

In some embodiments, this disclosure provides methods of treating apatient having an inflammatory disorder comprising administering to thepatient an effective amount of a pharmaceutical composition comprising anNIF, or a pharmaceutically acceptable salt of a nNIF, and apharmaceutically acceptable carrier to reduce a pathological effect orsymptom of the inflammatory disorder.

In other embodiments, the disclosure provides methods of treating apatient having an inflammatory disorder comprising administering to thepatient an effective amount of a pharmaceutical composition comprising anNIF analog, or a pharmaceutically acceptable salt of a nNIF analog, anda pharmaceutically acceptable carrier to reduce a pathological effect orsymptom of the inflammatory disorder.

In yet other embodiments, the disclosure provides methods of treating apatient having an inflammatory disorder comprising administering to thepatient an effective amount of a pharmaceutical composition comprising anNRP, or a pharmaceutically acceptable salt of a nNRP, and apharmaceutically acceptable carrier to reduce a pathological effect orsymptom of the inflammatory disorder.

In still other embodiments, the disclosure provides methods of treatinga patient having an inflammatory disorder comprising administering tothe patient an effective amount of a pharmaceutical compositioncomprising an nNRP analog, or a pharmaceutically acceptable salt of anNRP analog, and a pharmaceutically acceptable carrier to reduce apathological effect or symptom of the inflammatory disorder.

In some embodiments, the patient may be a mammal. In certain embodimentsthe patient may be a human. Any patient or subject requiring inhibitionof NETosis and/or NET formation may potentially be a candidate fortreatment with a NIP, a pharmaceutically acceptable salt of a NIP, anNIF, a pharmaceutically acceptable salt of a nNIF, a nNIF analog, apharmaceutically acceptable salt of a nNIF analog, a nNRP, apharmaceutically acceptable salt of a nNRP, a nNRP analog, and/or apharmaceutically acceptable salt of a nNRP analog.

In some embodiments, the inflammatory disorder may at least partiallyinvolve or be at partially caused by neutrophil extracellular trap (NET)formation and/or NETosis. In some embodiments, the inflammatory disordermay be an acute inflammatory disorder, a chronic inflammatory disorder,and/or an immune disorder. In other embodiments, the inflammatorydisorder may be an autoimmunity disorder. In yet other embodiments, theinflammatory disorder may be a disorder of coagulation.

In some embodiments, the inflammatory disorder may be one or more of,but not limited to, acute respiratory distress syndrome (ARDS),bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease(COPD), cystic fibrosis, inflammation in cancer and its complications,inflammatory bowel disease (IBD), inflammatory lung disease (ILD),influenza-induced pneumonitis, necrotizing enterocolitis (NEC), neonatalchronic lung disease (CLD), periodontitis, pre-eclampsia, retinopathy ofprematurity (ROP), sepsis, systemic inflammatory response syndrome(SIRS), thrombosis, transfusion-related acute lung injury (TRALI),vasculitis, autoimmune syndromes including, but not limited to,rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), andWegener's granulomatosis (WG), and disorders of nonresolvedinflammation. There are other inflammatory disorders not listed hereinbut known to those skilled in the art. For example, see Kumar et al.,Robbins and Cotran Pathologic Basis of Disease, pp. 43-77, 8th Edition,2010, Saunders Elsevier, Philadelphia, Pa.; Nathan, Nature, 2002, 420:846-852; and Amulic et al., Annu Rev Immunol, 2012, 30: 459-489.

In some embodiments, the pharmaceutical composition may substantiallyinhibit NET formation and/or NETosis. In other embodiments, thepharmaceutical composition may inhibit or substantially inhibitNET-mediated inflammatory tissue damage.

The particular form of NIP, nNIF, nNIF analog, nNRP, nNRP analog, and/orsalt thereof selected for inhibiting NETosis and/or NET formation can beprepared by a variety of techniques known for generating peptideproducts. For example, vertebrate forms of nNIF and nNRP can be obtainedby extraction from the natural source, using an appropriate combinationof protein isolation techniques. Other techniques are also within thescope of this disclosure.

In certain embodiments, NIPs, nNIFs, nNIF analogs, nNRPs, nNRP analogs,and/or salts thereof can be synthesized using standard techniques ofpeptide chemistry and can be assessed for inhibition of NETosis and/orNET formation activity. With respect to synthesis, the selected NIP,nNIF, nNIF analog, nNRP, nNRP analog, and/or salt thereof can beprepared by a variety of techniques for generating peptide products.Those NIPs, nNIFs, nNIF analogs, nNRPs, nNRP analogs, and/or saltsthereof that incorporate only L-amino acids can be produced incommercial quantities by application of recombinant DNA technology. Forthis purpose, DNA coding for the desired NIP, nNIF, nNIF analog, nNRP,and/or nNRP analog is incorporated into an expression vector andtransformed into a host cell (e.g., yeast, bacteria, or a mammaliancell) which is then cultured under conditions appropriate for NIP, nNIF,nNIF analog, nNRP, and/or nNRP analog expression. A variety of geneexpression systems have been adapted for this purpose, and typicallydrive expression of the desired gene from expression regulatory elementsused naturally by the chosen host.

In an approach applicable to the production of a selected NIP, nNIF,nNIF analog, nNRP, and/or nNRP analog, and one that may be used toproduce a NIP, nNIF, nNIF analog, nNRP, and/or nNRP analog thatincorporates non-genetically encoded amino acids and N- and C-terminallyderivatized forms, the techniques of automated peptide synthesis may beemployed, general descriptions of which appear, for example, in Stewartand Young, Solid Phase Peptide Synthesis, 2nd Edition, 1984, PierceChemical Company, Rockford, Ill.; Bodanszky and Bodanszky, The Practiceof Peptide Synthesis, 1984, Springer-Verlag, New York, N.Y.; and AppliedBiosystems 430A Users Manual, 1987, ABI Inc., Foster City, Calif. Inthese techniques, a NIP, nNIF, nNIF analog, nNRP, and/or nNRP analog isgrown from its C-terminal, resin-conjugated residue by the sequentialaddition of appropriately protected amino acids, using either the9-fluoroenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (t-Boc)protocols, as described for instance by Orskov et al., FEBS Lett, 1989,247(2): 193-196.

Once the desired NIP, nNIF, nNIF analog, nNRP, and/or nNRP analog hasbeen synthesized, cleaved from the resin and fully deprotected, thepeptide may then be purified to ensure the recovery of a singleoligopeptide having the selected amino acid sequence. Purification maybe achieved using any of the standard approaches, which include, but arenot limited to, reversed-phase high-pressure liquid chromatography(RP-HPLC) on alkylated silica columns (e.g., C4-, C8-, or C18-silica).Such column fractionation is generally accomplished by running lineargradients (e.g., 10-90%) of increasing percent organic solvent (e.g.,acetonitrile, in aqueous buffer) usually containing a small amount(e.g., 0.1%) of pairing agent such as trifluoroacetic acid (TFA) ortriethanolamine (TEA). Alternatively, ion-exchange HPLC can be employedto separate peptide species on the basis of their chargecharacteristics. Column fractions are collected, and those containingpeptide of the desired and/or required purity are optionally pooled. Inone embodiment of the invention, the NIP, nNIF, nNIF analog, nNRP,and/or nNRP analog may then be treated in the established manner toexchange the cleavage acid (e.g., TFA) with a pharmaceuticallyacceptable acid, such as acetic, hydrochloric, phosphoric, maleic,tartaric, succinic, and the like, to generate a pharmaceuticallyacceptable acid addition salt of the peptide.

Analogs of human NIPs, nNIFs, and/or nNRPs can be generated usingstandard techniques of peptide chemistry and can be assessed forinhibition of NETosis and/or NET formation activity, all according tothe guidance provided herein. Particularly preferred analogs of theinvention are those based upon the sequences of human nNIF (SEQ IDNO: 1) and/or CRISPP (SEQ ID NO: 2), as follows (wherein X can be anynaturally occurring amino acid):

SEQ ID NO: 1 KAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK SEQ ID NO: 2 MXIPPEVKFNKPFVFLMIDQNTKVPLFMGK

Any substitution, addition, or deletion of an amino acid or amino acidsof a NIP, nNIF, and/or nNRP that does not destroy the NET-inhibitoryactivity of the NIP, nNIF, and/or nNRP may be usefully employed in thisdisclosure. In certain embodiments, the NIP, nNIF, and/or nNRP analogsare at least as active as the native human NIP, nNIF, and/or nNRP.NET-inhibitory activity may be determined in vitro as described in thisdisclosure. In other embodiments, the NIP, nNIF, and/or nNRP analog hasone or more enhanced properties compared with the native human NIP,nNIF, and/or nNRP. For example, such analogs may exhibit enhanced serumstability, enhanced receptor binding and enhanced signal-transducingactivity. Other modifications to NIPs, nNIFs, nNIF analogs, nNRPs,and/or nNRP analogs that may usefully be employed in this disclosure arethose which render the molecule resistant to oxidation.

A researcher may determine whether a particular NIP, nNIF, nNIF analog,nNRP, nNRP analog, and/or salt thereof may be used to treat aninflammatory disorder by administering the peptide or analog toindividuals who have the inflammatory disorder. The researcher may thendetermine, using diagnostic biomarkers, whether the individuals thustreated show decreased inflammation and improvement of the inflammatorycondition.

The disclosure also encompasses non-conservative substitutions of aminoacids in any vertebrate NIP, nNIF, and/or nNRP sequence, provided thatthe non-conservative substitutions occur at amino acid positions knownto vary in NIPs, nNIFs, and/or nNRPs isolated from different species.Non-conserved residue positions are readily determined by aligning knownvertebrate NIP, nNIF, and/or nNRP sequences.

For administration to patients, the NIP, nNIF, nNIF analog, nNRP, nNRPpeptide, and/or salt thereof, may be provided in pharmaceuticallyacceptable form (e.g., as a preparation that is sterile-filtered, e.g.,through a 0.22μ filter, and substantially pyrogen-free). It may bedesired that the NIP, nNIF, and/or nNRP peptide to be formulatedmigrates as a single or individualized peak on HPLC, exhibits uniformand authentic amino acid composition and sequence upon analysis thereof,and otherwise meets standards set by the various national bodies whichregulate quality of pharmaceutical products.

The aqueous carrier or vehicle may be supplemented for use as aninjectable with an amount of gelatin that serves to depot the NIP, nNIF,nNIF analog, nNRP, nNRP analog, and/or salt thereof at or near the siteof injection, for its slow release to the desired site of action.Concentrations of gelatin effective to achieve the depot effect areexpected to lie in the range from 10-20%. Alternative gelling agents,such as hyaluronic acid (HA), may also be useful as depoting agents.

The NIPs, nNIFs, nNIF analogs, nNRPs, nNRP analogs, and/or salts thereofof the present disclosure may also be formulated as slow releaseimplantation formulations for extended and sustained administration ofthe NIP, nNIF, nNIF analog, nNRP, nNRP analog, and/or salt thereof.Examples of such sustained release formulations include composites ofbiocompatible polymers, such as poly(lactic acid),poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid,collagen, and the like. The structure, selection, and use of degradablepolymers in drug delivery vehicles have been reviewed in severalpublications, including, Domb et al., Polym Advan Technol, 1992, 3:279-292. Additional guidance in selecting and using polymers inpharmaceutical formulations can be found in the text by Chasin andLanger (eds.), Biodegradable Polymers as Drug Delivery Systems, Vol. 45of Dekker, Drugs and the Pharmaceutical Sciences, 1990, New York, N.Y.Liposomes may also be used to provide for the sustained release of anNIF, nNIF analog, nNRP, nNRP analog, and/or salt thereof. Detailsconcerning how to use and make liposomal formulations of drugs ofinterest can be found in, among other places, U.S. Pat. Nos. 4,944,948;5,008,050; 4,921,706; 4,927,637; 4,452,747; 4,016,100; 4,311,712;4,370,349; 4,372,949; 4,529,561; 5,009,956; 4,725,442; 4,737,323; and4,920,016. Sustained release formulations may be of particular interestwhen it is desirable to provide a high local concentration of a NIP,nNIF, nNIF analog, nNRP, nNRP analog, and/or salt thereof (e.g., nearthe site of inflammation to inhibit NETosis and/or NET formation, etc.).

The NIPs, nNIFs, nNIF analogs, nNRPs, nNRP analogs, and/or salts thereofof the present disclosure may also be incorporated into a device ordevices, both implanted or topical, for extended and sustainedadministration of the NIP, nNIF, nNIF analog, nNRP, nNRP analog, and/orsalt thereof.

For therapeutic use, the chosen NIP, nNIF, nNIF analog, nNRP, nNRPanalog, and/or salt thereof may be formulated with a carrier that ispharmaceutically acceptable and is appropriate for delivering thepeptide by the chosen route of administration. Suitable pharmaceuticallyacceptable carriers are those used conventionally with peptide-baseddrugs, such as diluents, excipients and the like. Reference may be madeto Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012,Pharmaceutical Press, London, UK. In one embodiment of the invention,the compounds may be formulated for administration by infusion or byinjection (e.g., subcutaneously, intramuscularly, or intravenously), andmay be accordingly utilized as aqueous solutions in sterile andpyrogen-free form and optionally buffered to physiologically tolerablepH (e.g., a slightly acidic or physiological pH). Thus, the compoundsmay be administered in a vehicle such as distilled water, saline,phosphate buffered saline, or 5% dextrose solution. Water solubility ofthe NIP, nNIF, nNIF analog, nNRP, nNRP analog, and/or salt thereof maybe enhanced, if desired, by incorporating a solubility enhancer, such asacetic acid.

Another aspect of the disclosure relates to methods of treatingcomplications of prematurity.

In embodiments, this disclosure provides for methods of treating apatient having a complication of prematurity comprising administering tothe patient an effective amount of a pharmaceutical compositioncomprising a NIP, or a pharmaceutically acceptable salt of a NIP, and apharmaceutically acceptable carrier to reduce a pathological effect orsymptom of the complication of prematurity, such as the prolonged needfor oxygen support associated with neonatal chronic lung disease or theneed for surgical intervention or prolonged total parenteral nutritionin infants that develop necrotizing enterocolitis.

In some embodiments, this disclosure provides for methods of treating apatient having a complication of prematurity comprising administering tothe patient an effective amount of a pharmaceutical compositioncomprising a nNIF, or a pharmaceutically acceptable salt of a nNIF, anda pharmaceutically acceptable carrier to reduce a pathological effect orsymptom of the complication of prematurity.

In other embodiments, the disclosure provides methods of treating apatient having a complication of prematurity comprising administering tothe patient an effective amount of a pharmaceutical compositioncomprising a nNIF analog, or a pharmaceutically acceptable salt of anNIF analog, and a pharmaceutically acceptable carrier to reduce apathological effect or symptom of the complication of prematurity.

In yet other embodiments, the disclosure provides methods of treating apatient having a complication of prematurity comprising administering tothe patient an effective amount of a pharmaceutical compositioncomprising a nNRP, or a pharmaceutically acceptable salt of a nNRP, anda pharmaceutically acceptable carrier to reduce a pathological effect orsymptom of the complication of prematurity.

In still other embodiments, the disclosure provides methods of treatinga patient having a complication of prematurity comprising administeringto the patient an effective amount of a pharmaceutical compositioncomprising a nNRP analog, or a pharmaceutically acceptable salt of anNRP analog, and a pharmaceutically acceptable carrier to reduce apathological effect or symptom of the complication of prematurity.

In some embodiments, the patient may be a mammal. In certain embodimentsthe patient may be a human.

In some embodiments, the complication of prematurity may at leastpartially involve or be at least partially caused by neutrophilextracellular trap (NET) formation and/or NETosis. In certainembodiments, the pharmaceutical composition may substantially inhibitNET formation and/or NETosis. In other embodiments, the pharmaceuticalcomposition may inhibit or substantially inhibit NET-mediatedinflammatory tissue damage.

In some embodiments, the complication of prematurity may be one or moreof, but not limited to, necrotizing enterocolitis (NEC), respiratorydistress syndrome (RDS), pneumonia, bronchopulmonary dysplasia (BPD),neonatal chronic lung disease (CLD), neurodevelopmental delay,retinopathy of prematurity (ROP), and/or sepsis.

A further aspect of the disclosure relates to methods of prophylaxisagainst inflammatory disorders.

In some embodiments, the disclosure provides for methods of prophylaxisagainst an inflammatory disorder in a patient at risk of developing aninflammatory disorder, comprising administering to the patient aneffective amount of a pharmaceutical composition comprising a NIP, or apharmaceutically acceptable salt of a NIP, and a pharmaceuticallyacceptable carrier to reduce the risk of developing a pathologicaleffect or symptom of the inflammatory disorder.

In some embodiments, the disclosure provides for methods of prophylaxisagainst an inflammatory disorder in a patient at risk of developing aninflammatory disorder, comprising administering to the patient aneffective amount of a pharmaceutical composition comprising a nNIF, or apharmaceutically acceptable salt of a nNIF, and a pharmaceuticallyacceptable carrier to reduce the risk of developing a pathologicaleffect or symptom of the inflammatory disorder.

In other embodiments, the disclosure provides for methods of prophylaxisagainst an inflammatory disorder in a patient at risk of developing aninflammatory disorder comprising administering to the patient aneffective amount of a pharmaceutical composition comprising a nNIFanalog, or a pharmaceutically acceptable salt of a nNIF analog, and apharmaceutically acceptable carrier to reduce the risk of developing apathological effect or symptom of the inflammatory disorder.

In yet other embodiments, the disclosure provides for methods ofprophylaxis against an inflammatory disorder in a patient at risk ofdeveloping an inflammatory disorder comprising administering to thepatient an effective amount of a pharmaceutical composition comprising anNRP, or a pharmaceutically acceptable salt of a nNRP, and apharmaceutically acceptable carrier to reduce the risk of developing apathological effect or symptom of the inflammatory disorder.

In still other embodiments, the disclosure provides for methods ofprophylaxis against an inflammatory disorder in a patient at risk ofdeveloping an inflammatory disorder comprising administering to thepatient an effective amount of a pharmaceutical composition comprising anNRP analog, or a pharmaceutically acceptable salt of the nNRP analog,and a pharmaceutically acceptable carrier to reduce the risk ofdeveloping a pathological effect or symptom of the inflammatorydisorder.

In some embodiments, the patient may be a mammal. In other embodimentsthe patient may be a human.

In some embodiments, the inflammatory disorder may at least partiallyinvolve or be at partially caused by neutrophil extracellular trap (NET)formation and/or NETosis. In some embodiments, the inflammatory disordermay be an acute inflammatory disorder. In other embodiments, theinflammatory disorder may be a chronic inflammatory disorder. In otherembodiments, the inflammatory disorder may be an autoimmunity disorder.In yet other embodiments, the inflammatory disorder may be a disorder ofcoagulation.

In some embodiments, the inflammatory disorder may be one or more of,but not limited to, the inflammatory disorders defined and/or listedabove.

In some embodiments, the pharmaceutical composition may substantiallyinhibit NET formation and/or NETosis. In other embodiments, thepharmaceutical composition may inhibit or substantially inhibitNET-mediated inflammatory tissue damage.

Another aspect of the disclosure relates to methods of prophylaxisagainst complications of prematurity.

In embodiments, this disclosure provides methods of prophylaxis againstcomplications of prematurity in a patient at risk of developing acomplication of prematurity comprising administering to the patient aneffective amount of a pharmaceutical composition comprising a neonatalNIP, or a pharmaceutically acceptable salt of a NIP, and apharmaceutically acceptable carrier to reduce the risk of developing apathological effect or symptom of the complication of prematurity.

In some embodiments, this disclosure provides methods of prophylaxisagainst complications of prematurity in a patient at risk of developinga complication of prematurity comprising administering to the patient aneffective amount of a pharmaceutical composition comprising a neonatalnNIF, or a pharmaceutically acceptable salt of a nNIF, and apharmaceutically acceptable carrier to reduce the risk of developing apathological effect or symptom of the complication of prematurity.

In certain embodiments, the disclosure provides methods of prophylaxisagainst complications of prematurity in a patient at risk of developinga complication of prematurity, comprising administering to the patientan effective amount of a pharmaceutical composition comprising a nNIFanalog, or a pharmaceutically acceptable salt of a nNIF analog, and apharmaceutically acceptable carrier to reduce the risk of developing apathological effect or symptom of the complication of prematurity.

In yet other embodiments, the disclosure provides methods of prophylaxisagainst complications of prematurity in a patient at risk of developinga complication of prematurity, comprising administering to the patientan effective amount of a pharmaceutical composition comprising anNIF-Related Peptide (nNRP), or a pharmaceutically acceptable salt of anNRP, and a pharmaceutically acceptable carrier to reduce the risk ofdeveloping a pathological effect or symptom of the complication ofprematurity.

In still other embodiments, the disclosure provides methods ofprophylaxis against complications of prematurity in a patient at risk ofdeveloping a complication of prematurity, comprising administering tothe patient an effective amount of a pharmaceutical compositioncomprising a nNRP analog, or a pharmaceutically acceptable salt of anNRP analog, and a pharmaceutically acceptable carrier to reduce therisk of developing a pathological effect or symptom of the complicationof prematurity.

In some embodiments, the patient may be a mammal. In other embodimentsthe patient may be a human.

In some embodiments, the complication of prematurity may at leastpartially involve or be at least partially caused by neutrophilextracellular trap (NET) formation and/or NETosis. In other embodiments,the pharmaceutical composition may substantially inhibit NET formationand/or NETosis. In yet other embodiments, the pharmaceutical compositionmay inhibit or substantially inhibit NET-mediated inflammatory tissuedamage.

In other embodiments, the complication of prematurity may be one or moreof, but not limited to, necrotizing enterocolitis (NEC), respiratorydistress syndrome (RDS), pneumonia, bronchopulmonary dysplasia (BPD),neonatal chronic lung disease (CLD), neurodevelopmental delay,retinopathy of prematurity (ROP), and/or sepsis.

Pharmaceutical Compositions

In a further aspect, this disclosure relates to pharmaceuticalcompositions comprising NIPs.

In some embodiments, the pharmaceutical composition may comprise aneonatal NET-Inhibitory Factor (nNIF), or a pharmaceutically acceptablesalt of a nNIF, and a pharmaceutically acceptable carrier. In anotherembodiment, the pharmaceutical composition may comprise a nNIF analog,or a pharmaceutically acceptable salt of a nNIF analog, and apharmaceutically acceptable carrier. In yet other embodiments, thepharmaceutical composition may comprise a nNIF-Related Peptide (nNRP),or a pharmaceutically acceptable salt of a nNRP, and a pharmaceuticallyacceptable carrier. In still other embodiments, the pharmaceuticalcomposition may comprise a nNRP analog, or a pharmaceutically acceptablesalt of a nNRP analog, and a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may comprisenNIF, or the salt thereof, and the nNIF, or the salt thereof, maycomprise the amino acid sequence:

SEQ ID NO: 1: KAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

In other embodiments, at least one amino acid of nNIF, the salt of thenNIF, the nNIF analog, the salt of the nNIF analog, the nNRP, the saltof the nNRP, the nNRP analog, or the salt of the nNRP analog, may bebound to a chemical modifier. In some embodiments, the chemical modifiermay be selected from at least one of a lipid, a polyethylene glycol(PEG), a saccharide, or any other suitable molecule. Other chemicalmodifications of the pharmaceutical composition, for example,cationization, methylization, and cyclization, are also within the scopeof this disclosure.

Attachment of a lipid to the peptide (lipidization) may increaselipophilicity of the pharmaceutical composition.

Attachment of a PEG to the peptide (PEGylation) increases the molecularweight of the peptide. In some embodiments, PEGylation may improvesolubility of the pharmaceutical composition. In other embodiments,PEGylation may reduce dosage frequency and/or toxicity of thepharmaceutical composition. In other embodiments, PEGylation may extendcirculating life of the pharmaceutical composition, and/or extendstability of the pharmaceutical composition, and/or may enhanceprotection of the pharmaceutical composition from proteolyticdegradation. PEGylation may also reduce immunogenicity and/orantigenicity of the pharmaceutical composition.

Attachment of one or more saccharides to the peptide (glycosylation) mayserve a variety of functional and/or structural roles in thepharmaceutical composition. Glycosylation may improve delivery of thepharmaceutical composition to a target or to targets of choice.Glycosylation may also reduce the toxicity of the pharmaceuticalcomposition.

In some embodiments, the pharmaceutical composition comprising nNIF, thesalt of the nNIF, the nNIF analog, the salt of the nNIF analog, thenNRP, the salt of the nNRP, the nNRP analog, or the salt of the nNRPanalog, may be present in an amount effective to inhibit, or tosubstantially inhibit, damage selected from at least one of inflammatorytissue injury and/or inflammatory vascular injury.

In some embodiments, the pharmaceutical composition comprising nNIF, thesalt of the nNIF, the nNIF analog, the salt of the nNIF analog, thenNRP, the salt of the nNRP, the nNRP analog, or the salt of the nNRPanalog, may not globally depress functions of polymorphonuclearleukocytes (PMNs). The functions of PMNs include, but are not limitedto, chemotaxis, phagocytosis, reactive oxygen species (ROS) generation,cytokine/chemokine synthesis and secretion, NET formation/NETosis,and/or intracellular/extracellular bacterial killing. In certainembodiments, the pharmaceutical composition may not inhibit orsubstantially inhibit PMN phagocytosis. In other embodiments, thepharmaceutical composition may not inhibit or substantially inhibit PMNchemotaxis. In yet other embodiments, the pharmaceutical composition maynot inhibit or substantially inhibit generation of ROS. In otherembodiments, the pharmaceutical composition may not inhibit orsubstantially inhibit PMN intracellular bacterial killing.

In some embodiments, the pharmaceutical composition may comprise a nNIFanalog, a salt of a nNIF analog, a nNRP analog, or a salt of a nNRPanalog, and the analog or the salt of the analog may not be a naturallyoccurring analog or salt of the analog.

In some embodiments, the pharmaceutical composition may be present in anamount effective to inhibit, or to substantially inhibit, NET formationand/or NETosis. In some embodiments, the NET formation and/or NETosismay be stimulated by a bacterium, a fungus, a parasite, a virus, and/orany other appropriate stimulator of NET formation and/or NETosis. Incertain embodiments, the virus may be a hemorrhagic fever virus.Hemorrhagic fever viruses are described, e.g., in Borio et al., JAMA,2002, 287(18): 2391-2405 and include, but are not limited to,filoviruses such as Ebola virus and Marburg virus, arenaviruses such asLassa virus, hantaviruses, and flaviviruses such as dengue virus andyellow fever virus. In other embodiments, the NET formation and/orNETosis may be stimulated by one or more bacterial species, including,but not limited to, Bacillus species, Escherichia species, Francisellaspecies, Streptococcus species, Staphylococcus species, Yersiniaspecies, and/or any other appropriate gram-negative or gram-positivebacterium or bacteria. In embodiments, the Bacillus species may beBacillus anthracis (anthrax). In embodiments, the Escherichia speciesmay be Escherichia coli. In embodiments, the Francisella species may beFrancisella tularensis (tularemia). In embodiments, the Staphylococcusspecies may be Staphylococcus aureus.

In other embodiments, the NET formation and/or NETosis may be stimulatedby beta-defensin 1, HIV-1, lipopolysaccharide (LPS), phorbol myristateacetate (PMA), and/or Staphylococcus aureus alpha-toxin.

In some embodiments, the pharmaceutical composition may comprise nNRP,Cancer-Associated SCM-Recognition, Immune Defense Suppression, andSerine Protease Protection Peptide (CRISPP), and/or a CRISPP analog. Insome other embodiments, the pharmaceutical composition may compriseA1ATm³⁵⁸, and/or an A1ATm³⁵⁸ analog, as A1ATm³⁵⁸ also inhibits NETformation. In other embodiments, the pharmaceutical may comprise anothernNRP. In other embodiments, the nNRP may be an isolated and purifiedcomponent of umbilical cord blood.

In an additional aspect, this disclosure relates to compositions forinhibiting the formation of NETs and/or NETosis in a mammal.

In some embodiments, a composition for inhibiting the formation of NETsand/or NETosis in a mammal may comprise an nNIF, a pharmaceuticallyacceptable salt of the nNIF, a nNIF analog, a pharmaceuticallyacceptable salt of the nNIF analog, an nNRP, a pharmaceuticallyacceptable salt of the nNRP, a nNRP analog, or a pharmaceuticallyacceptable salt of the nNRP analog, and a pharmaceutically acceptablecarrier. In certain embodiments the mammal may be a human.

Isolated and Purified Peptides

In a further aspect, this disclosure relates to a NET-inhibitory peptide(NIP).

In some embodiments, the NIP may be an isolated and purified nNIFprotein comprising SEQ ID NO: 1. In certain other embodiments, theisolated and purified nNIF protein may comprise at least twenty-fourcontiguous amino acids of SEQ ID NO: 1. In yet other embodiments, theisolated and purified nNIF protein may comprise at least twelvecontiguous amino acids of SEQ ID NO: 1. In still other embodiments, theisolated and purified nNIF protein may comprise at least six contiguousamino acids of SEQ ID NO: 1.

In some embodiments, the NIP may be an isolated and purified nNIFprotein wherein the sequence may be at least eighty percent identical toSEQ ID NO: 1. In other embodiments, the isolated and purified nNIF maybe at least sixty percent identical to SEQ ID NO: 1. In yet otherembodiments, the isolated and purified nNIF may be at least fortypercent identical to SEQ ID NO: 1. In still other embodiments, theisolated and purified nNIF may be at least twenty percent identical toSEQ ID NO: 1.

In another aspect, this disclosure relates to a nNIF protein analog. Insome embodiments, the nNIF protein analog may be an isolated andpurified nNIF analog.

An effective dosage and treatment protocol may be determined byconventional means, e.g., by starting with a low dose in laboratoryanimals and then increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Primary among these is whether any NIPs arenormally circulating in the plasma and, if so, the amount of any suchNIPs. Additional factors include the size of the patient, the age of thepatient, the general condition of the patient, the particular disorderbeing treated, the severity of the disorder, the presence of other drugsin the patient, the in vivo activity of the NIP, nNIF, nNIF analog,nNRP, nNRP analog, salt thereof, and the like. The trial dosages may bechosen after consideration of the results of animal studies and theclinical literature.

There are many specific therapeutic regimens used to assess whether amolecule has a desired effect. A researcher faced with the task ofdetermining whether a particular NIP, nNIF, nNIF analog, nNRP, and/ornNRP analog may be used for inhibition of NETosis and/or NET formationwould choose the appropriate regimen to make this determination.

Delivery methods and formulations useful for administering peptides toindividuals are known in the art, and a skilled person would be able todetermine the suitability of any particular method of delivery of apeptide to an individual for particular circumstances. For the purposesof illustration only, the following examples of methods and formulationsfor administering peptides to individuals are provided.

Peptides may be administered to individuals orally; however, actions ofthe digestive system may reduce the bioavailability of the peptide. Inorder to increase peptide oral bioavailability, peptides may beadministered in formulations containing enzyme inhibitors, or thepeptides may be administered as part of a micelle, nanoparticle, oremulsion in order to protect the peptide from digestive activity.

Peptides may also be administered by means of an injection. The peptidesmay be injected subcutaneously, intramuscularly, or intravenously.Further disclosure regarding methods of administering peptides throughinjection is found, e.g., in U.S. Pat. No. 5,952,301.

Peptides may further be administered by pulmonary delivery. A dry powderinhalation system may be used, wherein peptides are absorbed through thetissue of the lungs, allowing delivery without injection, whilebypassing the potential reduction in bioavailability seen with oraladministration. See Onoue et al., Expert Opin Ther Pat, 2008, 18: 429.

For use in inhibiting NETosis and/or NET formation in a mammal,including a human, the present disclosure provides in one of its aspectsa package or kit, in the form of a sterile-filled vial or ampoule, thatcontains a NETosis and/or NET formation inhibiting amount of a NIP,nNIF, nNIF analog, nNRP, nNRP analog, and/or salt thereof in either unitdose or multi-dose amounts, wherein the package or kit incorporates alabel instructing use of its contents for the inhibition of such NETosisand/or NET formation. In one embodiment of the invention, the package orkit contains the NIP, nNIF, nNIF analog, nNRP, nNRP analog, and/or saltthereof and the desired carrier, as an administration-ready formulation.Alternatively, and according to another embodiment of the invention, thepackage or kit provides the NIP, nNIF, nNIF analog, nNRP, nNRP analog,and/or salt thereof, in a form, such as a lyophilized form, suitable forreconstitution in a suitable carrier, such as phosphate-buffered saline.

In one embodiment, the package or kit is a sterile-filled vial orampoule containing an injectable solution which comprises an effective,NETosis and/or NET formation inhibiting amount of NIP, nNIF, nNIFanalog, nNRP, nNRP analog and/or salt thereof dissolved in an aqueousvehicle.

EXAMPLES

To further illustrate these embodiments, the following examples areprovided. These examples are not intended to limit the scope of theclaimed invention, which should be determined solely on the basis of theattached claims.

Example 1—Comparing Human Adult and Neonatal PMNs

NET-formation was assessed in human and neonatal PMNs. In contrast toadult human PMNs, as shown in FIG. 1B, neonatal PMNs isolated from cordblood do not form NETs. Further, NET-mediated bacterial killing wasassessed in human adult and neonatal PMNs. As indicated in FIG. 1C,neonatal PMNs demonstrate significantly decreased total and NET-mediatedbacterial killing as compared to adult PMNs. * p<0.05, ** p<0.001.

Example 2—Inducing NET Formation by S. aureus Sepsis

NET-formation was assessed in control and stimulated human PMNs at a 1hour time point. Human PMNs were stimulated with either LPS, controlplasma, or with plasma isolated from patients with S. aureus sepsis.Plasma from patients with S. aureus sepsis induces NET formation byhuman PMNs, as shown in FIGS. 2A-2C.

Example 3—Observing Impaired NET Formation at Birth

PMNs were isolated from the cord blood of infants born at less than 30weeks gestation and, subsequently, from serially drawn peripheral bloodfrom the same infants through the first 60 days of life. As shown inFIGS. 3A-3C, NET formation was assessed qualitatively andquantitatively, and impaired NET formation by PMNs was observed at thetime of birth. In contrast, robust NET formation by PMNs was observedfrom blood samples isolated on day of life 3 or later.

Example 4—Demonstrating NET Formation Inhibitor in Cord Blood

PMNs from healthy adult donors and from 28 day old preterm infants werepre-incubated for one hour with thawed cord blood plasma from thatparticular preterm infant or with autologous plasma collected that dayfrom either the preterm infant peripheral blood sample or the bloodsample from the healthy adult. NET formation was then assessed in vitrofollowing LPS-stimulation (100 ng/mL) for 2 hours. Cord blood plasmapre-incubation significantly decreased NET formation by both neonataland adult LPS-stimulated PMNs as compared to PMNs pre-incubated withcontrol plasma (see FIGS. 3B and 14C).

As illustrated in FIGS. 3B and 14C, human cord blood pre-incubationexperiments demonstrate that cord blood plasma can inhibit NET formationby day of life 28 autologous preterm infant PMNs and by healthy adultdonor PMNs, while pre-incubation with autologous plasma isolated fromthe respective study subjects on that day may not. FIG. 3B showsNET-associated, extracellular DNA and nuclear DNA (60× magnification).

Extracellular histone H₃ release was used as a quantifiable surrogatefor NET formation as depicted in FIG. 14C. As illustrated, extracellularhistone content (fold change over baseline) is represented on they-axis, and data from five preterm infant's PMNs on day of life 28 andfive adult's PMNs, from the above-described plasma switch experiments,are represented on the x-axis. * denotes p<0.05 LPS/Adult versusLPS/Preterm, † denotes p<0.01 LPS/Preterm versus cord blood LPS/Preterm,and † denotes p<0.001 LPS/Adult versus cord blood LPS/Adult. The one-wayANOVA statistical tool with Tukey's post-hoc testing was employed.

Cord blood and peripheral blood plasma switch experiments demonstratedthat autologous cord blood plasma inhibits NET formation byNET-competent PMNs isolated from the same infant on day of life 28, andalso by PMNs isolated from healthy adults, indicating that an inhibitoryfactor that blocks PMN NET formation is present in cord blood, but thatactivity of the factor disappears or is dramatically reduced after birth(see FIGS. 3B and 3C).

Example 5—Identifying Neonatal NET-Inhibitory Factor (nNIF)

Proteomic techniques were used to compare the cord blood plasma proteomewith that of plasma isolated from the same preterm infant at 28 days ofage (see FIGS. 3B and 14C). Following abundant protein removal, parallel2-dimensional gel electrophoresis was performed on each plasma sample,separating proteins first by isoelectric focusing and then by molecularweight. Six protein spots were noted to be differentially present uponcomparison of the two gels. Four protein clusters were present in thegel of cord blood plasma but not in the 28 day plasma gel; two proteinclusters were present in the 28 day plasma gel but not the cord bloodplasma gel. Each of these protein clusters were cut out of therespective gels for protein identification. The oligopeptide sequencesfollowing trypsin-digest and tandem mass spectroscopy were compared tothe NCBI Human trypsin-specific database (current through May 13, 2011).A partial list of proteins identified by mass spectroscopy on one of theprotein clusters present on the cord blood gel but not on the 28 dayplasma gel is included in FIG. 4A. Of the proteins identified in thisprotein cluster, two related peptides with predicted molecular weightsof ≈4 kD and protein scores of >100 became candidate peptides forNET-inhibitory activity. The novel peptide was named neonatalNET-inhibitory Factor (nNIF). A nNIF-related peptide (NRP) was known inthe literature: Cancer-Associated SCM-Recognition, ImmunedefenseSuppression, and Serine Protease Protection Peptide (CRISPP). Next, itwas determined that nNIF and CRISPP have significant homology to thecarboxy terminus of full length alpha 1-antitrypsin (AAT, also referredto herein as A1AT) (see FIG. 4C). Using a polyclonal antibody generatedagainst the last 18 amino acids of AAT, it was demonstrated usingwestern blotting that nNIF is present in increased amounts in cord bloodplasma as compared to healthy adult plasma (see FIGS. 15A and 15B).

As discussed, protein expression in cord blood plasma and day of life 28plasma from the same preterm infant was compared using 2D-gelelectrophoresis, and a protein spot present in cord blood but not in dayof life 28 plasma was detected. A list of nNIF candidate proteins fromthat protein spot following mass spectroscopy is outlined with expectedmolecular masses and protein scores in FIG. 4A. The list includes fulllength AAT and two 29 amino-acid peptides with significant homology tothe carboxy-terminus of AAT: nNIF and CRISPP. As illustrated in FIG. 4C,nNIF and AAT share significant carboxy terminus homology. The sequencesobtained from mass spectroscopy are shown and compared, and thepublished sequence of CRISPP is also compared. Western blotting using apolyclonal antibody against the carboxy-terminus of AAT is shown in FIG.15A, demonstrating increased expression of nNIF (≈4-6 kD) in cord bloodas compared to adult plasma. As shown in FIG. 15B, these results werequantified using relative fluorescence and a statistically significantdifference was found between four cord blood plasma nNIF levels (CB) andthose of four healthy adult controls (A). * denotes p<0.05. TheStudent's t-test statistical tool was used.

In summary, protein expression in cord blood and day of life 28 plasmafrom the same preterm infant was compared using 2D-gel electrophoresis.A protein spot present in cord blood but not in day of life 28 plasmawas detected. An endogenous peptide (≈4-6 kD) with significant NETinhibitory activity was identified. This peptide was provisionally namedneonatal NET-inhibitory factor (nNIF).

Example 6—Characterizing the nNIF Peptide

Mass spectroscopy was used to further characterize the nNIF peptide ofExample 5 and to resolve major portions of its sequence. A list of nNIFcandidate proteins from the protein spot of Example 5 following massspectroscopy is outlined with expected molecular mass and protein scorein FIG. 4A. As shown in FIG. 4C, nNIF was found to have significanthomology to a carboxy-terminus cleavage fragment of A1AT. An antibodyagainst the carboxy terminus of A1AT was used to detect a 4-6 kD peptidein cord blood plasma that was only minimally present in adult plasma(see FIG. 4B).

Example 7—Further Characterization of the nNIF Peptide

Cord blood plasma was depleted of all full length and fragmented A1ATusing affinity purification (AP) with the A1AT carboxy-terminusantibody. As shown in FIGS. 5A-5F, cord blood plasma depleted of nNIFfailed to inhibit NET formation, while pretreatment with the AP elutedproteins inhibited NET formation by LPS-stimulated PMNs. Addition ofrecombinant full length A1AT to the depleted plasma did not inhibit NETformation. These data suggest that nNIF is a cleavage fragment of A1ATor a similar peptide and not the full length protein.

Example 8—Identifying the nNIF-Related Peptide (nNRP), CRISPP

Significant homology was identified between nNIF and Cancer-AssociatedSCM-Recognition, and Serine Protease Protection Peptide (CRISPP) (seeFIG. 4C). CRISPP peptide was synthesized and it was observed that CRISPPblocked NET formation by LPS-stimulated PMNs in a concentrationdependent manner. A scrambled control peptide did not block NETformation by LPS-stimulated PMNs.

Example 9—Inhibiting NET Formation with CRISPP Treatment

NET formation by PMA-stimulated adult PMNs with or without CRISPPpre-incubation was assessed. As shown in FIGS. 6A and 6B, CRISPPpretreatment (1 nM) for 1 hour prior to stimulation inhibited NETformation while pretreatment with a scrambled control peptide (1 nM) didnot. CRISPP was observed to block NET formation induced by PMA.

Example 10—Analyzing NET Formation in Multiple In Vitro Infection Models

A human histone H₃ release assay was used to quantify NET formation(n=2). NET-inhibitory activity of CRISPP was tested using in vitromodels of two human infectious diseases: Staphylococcus aureusbacteremia and dengue fever. S. aureus has previously been shown toinduce NET formation by PMNs from human adults. Pretreatment with CRISPPcompletely blocked NET formation by human PMNs incubated with live S.aureus (see FIGS. 7A and 7B). In contrast, incubation with a scrambledcontrol peptide did not block NET formation by human PMNs.

As shown in FIGS. 8A and 8B, human PMNs were incubated with dengue virus(0.05 MOI) and qualitatively assessed for NET formation. Mock incubationusing dengue virus media without infection served as a control. Denguevirus induced NET formation by PMNs from human adults. Pretreatment withCRISPP (1 nM) for 1 hour prior to stimulation completely blocked NETformation by human PMNs incubated with dengue virus. In contrast,incubation with a scrambled control peptide (1 nM) did not block NETformation by human PMNs.

Example 11—Determining Effect of CRISPP Treatment on PMN Functions

Total, phagocytotic, and NET-mediated extracellular bacterial killing ofa pathogenic strain of E. coli by human PMNs±LPS stimulation (100 ng/mL)was assessed, and the results are summarized in FIG. 9. PMNs were alsopretreated with CRISPP (1 nM), or a scrambled control peptide control (1nM). Phagocytotic killing was inhibited with cytochalasin B and Dpretreatment (10 μM). NET-mediated killing was inhibited using DNasetreatment to dismantle NETs prior to addition of the bacteria. CRISPPpretreatment significantly decreased total and NET-mediated bacterialkilling of E. coli, but did not alter phagocytic killing, as measuredusing the one-way ANOVA statistical tool with Newman-Keuls MultipleComparison Test post-hoc analysis.

Example 12—Assessing Effect of CRISPP Treatment in Murine Models

The ability of CRISPP to inhibit NET formation in vivo was assessedusing murine models of E. coli peritonitis. Outbred Swiss-Webster micewere injected intraperitoneally with 4.5×10⁷ colony-forming units (cfu)of E. coli±nNIF, CRISPP, or scrambled control peptide (SCR) (10 mg/kg,IP injection) one hour prior to E. coli infection. At the 3 hour timepoint after infection, 3 mice were sacrificed and the peritoneal fluidand peritoneal tissue were collected and analyzed for leukocyteaccumulation, bacteriology, and in vivo NET formation. Total mononuclearand polymorphonuclear leukocyte counts were determined in the peritonealfluid and demonstrated a statistically significant increase inperitoneal PMN accumulation in all experimental groups compared tocontrol (see FIG. 17A). Mice pretreated with CRISPP demonstrated asignificant increase in PMN accumulation compared to E. coli alone orSCR pretreated mice (see FIGS. 17A and 17B). This result appearsconsistent with a CRISPP-mediated decrease in neutrophil cell deaththrough inhibition of NETosis. See Fuchs et al., J Cell Biol, 2007, 176:231-241. The number of E. coli cfu/mL of peritoneal fluid in CRISPPpretreated mice was also determined compared to SCR mice (see FIG. 17C).A significant increase in peritoneal fluid bacterial concentration wasdetected in CRISPP pretreated animals compared to SCR, indicating thatthe inhibition of NETosis and NET formation may lead to decreasedbacterial killing and higher peritoneal fluid concentrations ofbacteria.

Survival of outbred Swiss mice was tracked in various treatment armsfollowing intraperitoneal injection of either LPS (20 mg/kg) or E. coli(4×10⁷ bacteria), as shown in FIGS. 10A and 10B. CRISPP treated mice (10mcg/kg/dose) received 2 doses intraperitoneally; one given 1 hour priorto infection and one dose given 6 hours after infection. The same dose,delivery route, and schedule were followed for the scrambled controlpeptide control mice. Six mice were assessed in both groups: LPS+CRISPPand LPS+scrambled control peptide. The LPS+CRISPP group survival wasstatistically greater than the LPS+scrambled control peptide group(p=0.02). Ten mice were assessed in each group: no treatment, E.coli+CRISPP, and E. coli+scrambled control peptide. No antibiotics weregiven. No deaths occurred in the no treatment group. The E. coli+CRISPPgroup survival was statistically greater than the E. coli+scrambledcontrol peptide group (p<0.0001). Referring to FIG. 11, CRISPP was alsoevaluated in a murine model of polymicrobial infection caused by cecalligation and puncture (CL/P). CL/P was also performed withoutconcomitant antibiotic treatment. Ten mice were assessed in each of theCL/P groups. The CL/P+CRISPP group survival approached statisticalsignificance compared with the CL/P control group (p=0.06). FIG. 20 is agraph depicting the assessment of ten mice in each of the followinggroups: no treatment, E. coli, CRISPP-Post/E. coli, and SCR-Post/E.coli. The survival increase seen in CRISPP-Post/E. coli mice compared toSCR-Post/E. coli mice approached statistical significance. For allexperiments in each of the three murine models, the Log-rank(Mantel-Cox) statistical tool was used to compare the survival curvesbetween groups and employed the post hoc Bonferroni correction. For allexperiments in all three models, * denotes p<0.05, ** denotes p<0.01,and *** denotes p<0.001.

Example 13—Analyzing NET Formation in CLD Using Human Tissue and a SheepModel

De-identified human lung tissue from preterm infants who died with CLDwas immunohistochemically analyzed for extracellular DNA and histone H₃,two key indicators of NET formation (see FIGS. 12A and 12B). Usingsimilar techniques, lung tissue samples collected from prematurely bornlambs in the sheep model of CLD were also examined. Lung tissue samplesfrom preterm lambs mechanically ventilated for 18-21 days demonstratedall of the hallmarks of CLD while control tissue samples taken fromlambs born either at term or prematurely but supported with CPAP alonedid not. Lung tissue samples from the mechanically ventilated pretermlambs demonstrated robust NET formation while that from control lambsdid not.

NET formation was assessed in paraffin-embedded human lung tissueobtained at autopsy of neonates who died with CLD. In a preliminaryanalysis, extracellular, alveolar histone H₃ accumulation consistentwith NET formation was found. NET formation was also assessed inparaffin-embedded preterm lamb lung tissue in the sheep model of CLD.Extracellular, alveolar NET formation was detected in the preterm lambCLD specimen but not in the control preterm lamb. Similar deposition ofNETs has been reported in a murine model of TRALI. Other hallmarks ofneonatal CLD in both human and lamb CLD samples, includinghypercellularity and alveolar simplification, were also observed.

Example 14—Characterizing NET Formation in Sheep PMNs

Referring to FIGS. 13A and 13B, PMNs from preterm lamb cord blood andfrom mature ewes were isolated. The isolated PMNs were stimulated withLPS (100 ng/mL) for 1 hour and NET formation was assessed qualitatively.Mature ewe PMNs were treated with CRISPP (1 nM) or a scrambled controlpeptide (1 nM) for 1 hour prior to LPS-stimulation (100 ng/mL) and NETformation was assessed qualitatively. PMNs isolated from lamb cord bloodfailed to form NETs in vitro, but PMNs isolated from mature sheeprobustly formed NETs following LPS stimulation. CRISPP pre-treatment ofsheep PMNs inhibited NET formation in a dose-dependent manner while thescrambled control peptide did not. CRISPP pre-treatment of sheep PMNsinhibited NET formation stimulated with PMA.

Example 15—Isolating PMNs

PMNs were isolated from acid-citrate-dextrose (ACD) orethylenediaminetetraacetic acid (EDTA) anticoagulated venous blood ofhealthy adults, healthy term infants, and prematurely born infants. Forthe 11 prematurely born infants from whom cord and peripheral bloodsamples were collected, cord and peripheral blood plasma and PMNpreparations were obtained at 5 separate time points throughout thefirst 2 months of life. PMN suspensions (>96% pure) were prepared bypositive immunoselection using anti-CD15-coated microbeads and anauto-MACS cell sorter (MILTENYI BIOTEC, INC.) and were resuspended at2×10⁶ cells/mL concentration in serum-free M-199 media at 37° C.

Table 1 (below) provides the demographics associated with the preterminfant cohort (n=11).

TABLE 1 Preterm Infant Demographic Information¹ Gestational ages atbirth 23⁶/₇-29⁰/₇ weeks Birth weight 570-1160 g Indication for pre-termdelivery Prolonged premature rupture of 8 membranes or preterm laborPregnancy induced hypertension 1 Placental abruption/preterm labor 0Bacterial blood culture results E. coli 0 Coagulase (−) Staphlococcus 2Group B Streptococcus 0 Negative 6 Meningitis 2 Pneumonia 2 Antibiotictreatment All treated, 2-14 d ¹Clinical characteristics and infectiouscomplications of preterm infant participants.

It was found that PMNs isolated from newborn infants, whether born atterm or prematurely, rapidly gained the ability to form NETs, asdemonstrated by qualitative and quantitative assays of NET formationfollowing in vitro stimulation with LPS (see FIGS. 14A and 14B; see alsoYost et al., Blood, 2009, 113: 6419-1154; and McInturff et al., Blood,2012, 120: 3118-3125). Robust NET formation was demonstrated as early asthe third day of life even for the most prematurely born infants and NETcompetency remained intact in PMNs isolated serially through two monthsof age.

Example 16—Isolating Platelets

Human platelets were isolated as described in Weyrich et al., J ClinInvest, 1996, 97: 1525-1534. Briefly, ACD anticoagulated whole blood wasspun at 100×g for 20 minutes. The platelet rich plasma (PRP) wascollected, transferred to a new 50 mL conical tube, and prostaglandin E1(PGE; 100 nM; CAYMAN CHEMICAL) was added. The PRP was then centrifugedat 1500 RPM for 20 minutes. After centrifugation, the supernatant wasremoved and the platelet pellet was resuspended with 10 mL of 37° C. PSGmedia and PGE (100 nM). Platelets were resuspended in serum-free M-199media to 1×10⁸ cells/mL with platelet counts acquired using a BECKMANCOULTER Particle Counter (BECKMAN COULTER).

Example 17—Live Cell Imaging of NET Formation

It was determined whether nNIF and CRISPP inhibit NET formation by humanPMNs using qualitative and quantitative assays of in vitro NETformation. Human PMNs isolated from healthy adult donors were stimulatedwith LPS (100 ng/mL) or phorbal-12-mystirate (PMA; 20 nM), ±nNIF (1 nM)or CRISPP (1 nM), pre-incubation (1 hour). Scrambled control peptide(SCR) generated using the identical amino acid content as CRISPP butlinked in a random order was used as a control. NET formation wasassessed 2 hours after stimulation. It was found that both nNIF andCRISPP significantly inhibited NET formation in the in vitro system ascompared to the SCR peptide control (see FIGS. 6A and 16A-16D).

NET formation was assessed by stimulated adult PMNs±nNIF or CRISPPpre-incubation, both qualitatively and quantitatively (see FIGS. 6A and16A-16D). nNIF or CRISPP pre-incubation (1 nM) for 1 hour prior tostimulation (LPS, 100 ng/mL or PMA, 20 nM) inhibited NET formation,while pretreatment with SCR (1 nM) did not appear to inhibit NETformation. The representative images of FIG. 16A (20× magnification) andFIG. 6A (60× magnification) show extracellular NET-associated DNA andnuclear DNA. The human histone H₃ release assay was used to quantify NETformation in stimulated PMNs pre-incubated±nNIF or CRISPP. As depictedin FIGS. 16B and 16C, extracellular histone content (fold change overbaseline) is represented on the y-axis, and the dashed line representsthe control values, arbitrarily set at 1. The data presented arerepresentative of 3 separate experiments performed using PMNs isolatedfrom 3 different participants. * denotes p<0.05 for CRISPP/PMA comparedto PMA and SCR/PMA groups, ** denotes p<0.05 for LPS and SCR/LPScompared to control, while † denotes p<0.05 for CRISPP/LPS and nNIF/LPScompared to both the LPS and SCR/LPS groups. The one-way ANOVAstatistical tool with Tukey's post-hoc testing was employed.

NET formation was assessed in the peritoneum using live cell imaging. Acell impermeable DNA dye stained all extracellular DNA, and a cellpermeable DNA dye served as a nuclear counterstain. A qualitativedecrease in NET formation in the peritoneal fluid of nNIF or CRISPPpretreated mice was found compared to the SCR control and a significantquantitative decrease in NET formation as assayed by histone H₃ releaseassay of the peritoneal fluid (see FIGS. 17D and 17E). NET formation wasalso examined on freshly resected peritoneal membrane tissue using livecell imaging. A qualitative and quantitative decrease in peritonealmembrane-associated NET formation was seen (see FIGS. 17F and 17G).These results demonstrate that nNIF and the NRP CRISPP can inhibit NETformation stimulated in in vivo as well as in vitro model systems.

The ability of CRISPP to inhibit NET formation induced by bacterial andviral pathogens was also tested. CRISPP inhibited NET formation inducedby incubation with S. aureus bacteria (MOI 100) and Dengue viralinfection (MOI 0.05) (see FIGS. 7A, 8A, and 8B; see also Example 18)demonstrating that nNIF can inhibit NET formation in reductionistexperimental models of bacterial and viral infection.

Qualitative assessment of NET formation was performed as previouslyreferenced in Yost et al., Blood, 2009, 113: 6419-6427 and McInturff etal., Blood, 2012, 120: 3118-3125. Briefly, primary PMNs isolated frompreterm infants, healthy term infants, and healthy adults (2×10⁶cells/mL) were incubated with control buffer or stimulated with LPS (100ng/mL), PMA (20 nM), or S. aureus bacteria (MOI 100) for 1 hour at 37°C. in 5% CO₂/95% air on glass coverslips coated with poly-L-lysine. Forselected experiments, primary PMNs were pre-incubated with autologousplasma, cord blood plasma, nNIF (1 nM), CRISPP (1 nM), or SCR (1 nM) for1 hour prior to imaging. After pre-incubation and/or stimulation, PMNswere gently washed with PBS and incubated with a mixture of cellpermeable (SYTO Green, MOLECULAR PROBES) and impermeable (SYTOX Orange,MOLECULAR PROBES) DNA fluorescent dyes. Confocal microscopy wasaccomplished using a FV300 1×81 Microscope and FLUOVIEW software(OLYMPUS). Both 20× and 60× objectives were used. Z-series images wereobtained at a step size 1 μm over a range of 20 μm for each field.OLYMPUS FLUOVIEW and ADOBE PHOTOSHOP CS2 software were used for imageprocessing.

Example 18—Imaging of Dengue Virus-Induced NET Formation

Primary PMNs isolated from healthy adults (2×10⁶ cells/mL) wereincubated with mock infection buffer or live dengue virus (MOI 0.05), asfor the live cell imaging discussed above. After a 1 hour incubation,the infected PMNs were fixed with 2% p-FA for 10 minutes prior toincubation with fluorescently-labeled, cell permeable and cellimpermeable DNA dyes, and imaged as for live cell imaging using confocalmicroscopy.

Example 19—Quantitating NET Formation and Supernatant Histone H₃ Content

Supernatant total histone H₃ content was determined as previouslyreferenced in McInturff et al., Blood, 2012, 120: 3118-3125. After livecell imaging of control and stimulated primary PMNs (2×10⁶/mL; variousagonists), the cells were incubated with PBS containing DNase (40 U/mL)for 15 minutes at room temperature to break down and release NETs formedin response to stimulation. The supernatant was gently removed andcentrifuged at 420×g for 5 minutes. The cell-free supernatant was thenmixed 1:3 with 4× Laemmli buffer prior to western blotting. A polyclonalprimary antibody against human histone H₃ protein (CELL SIGNALINGTECHNOLOGY) and infrared secondary antibodies (LI-COR BIOSCIENCES) wereused. Imaging and densitometry were performed on the ODYSSEY™ infraredimaging system (LI-COR BIOSCIENCES).

Example 20—Isolating and Identifying nNIF in Umbilical Cord Blood Plasma

Two plasma samples from a single preterm infant, one from the umbilicalcord blood, and one from a peripheral blood sample taken on day of life28, underwent abundant plasma protein removal (PROTEOSPIN, NORGEN) priorto 2D-electrophoresis using separation first by isoelectric focusing (pHrange 3-8) and then by size (TGX PRECAST GEL, BIORAD). The resultinggels were compared for differential protein content. Six differentiallyexpressed protein clusters were analyzed. Following trypsin digestionand tandem mass spectrometry using an LTQ-FT ion-trap/FTMS hybrid massspectrometer (THERMOELECTRON), candidate proteins/peptides wereidentified as potential NET-inhibitory substances.

Example 21—Affinity Purifying nNIF

To determine whether the 29 amino acid peptide identified in Example 5were the substances circulating in umbilical cord blood responsible forthe inhibition of NET formation, cord blood plasma was depleted of nNIFvia affinity purification using the carboxy-terminus AAT antibody. nNIFwas then eluted from the column and used in experiments to assess theNET-inhibitory activity of unaltered cord blood plasma, nNIF-depletedcord blood plasma, and affinity purified nNIF from cord blood plasma. Asa control, recombinant AAT in nNIF-depleted cord blood was also assessed(see FIGS. 5A-5F). PMNs isolated from healthy adult donors respondedwith robust NET formation following 2 hour incubation with LPS (100ng/mL). Pre-incubation with unaltered cord blood plasma inhibited invitro NET formation, consistent with earlier results (see FIGS. 3B and14C). Pre-incubation with nNIF-depleted cord blood plasma±rAAT (1 nM)did not. Pre-incubation with affinity purified nNIF, however, inhibitedNET formation by LPS-stimulated PMNs to a similar degree as theunaltered umbilical cord blood. Further experiments also showed thatfull-length, enzymatically active human AAT failed to inhibit NETformation by PMNs isolated from healthy adult donors while nNIF did (seeFIG. 15C). These results suggested that the 29 amino acid peptides nNIFand CRISPP detected in umbilical cord blood have NET-inhibitoryactivity. Synthesis of nNIF and CRISPP for in vitro studies of their NETinhibitory capacities was then performed.

As discussed above, following abundant plasma protein removal(PROTEOSPIN, NORGEN), a polyclonal antibody raised against the last 18amino acids of the carboxy terminus of full length AAT (LIFESPANBIOSCIENCES) was used to affinity purify nNIF from cord blood plasmausing an immunoprecipitation kit (THERMO SCIENTIFIC). Cord blood plasmadepleted of both nNIF and full length AAT was collected as were thepeptides captured by affinity purification for use in in vitro assays ofNET formation. Full length, enzymatically active AAT (SIGMA) was alsoresuspended in elution buffer and tested in parallel.

Example 22—Analyzing Peritoneal Fluid and Membranes

Animals were treated with CRISPP (10 mg/kg), nNIF (10 mg/kg), or SCR (10mg/kg) by i.p. injection 1 hour prior to infection (E. coli, 4.5×10⁷cfu/mouse, i.p. injection) or stimulation (LPS, 25 mg/kg, i.p. injection(SIGMA)). Control mice were injected with saline alone. The mice weresacrificed in a CO₂ chamber 3 hours post-infection/stimulation with theperitoneal contents harvested and analyzed. Briefly, the skin of theabdomen was cut open in the midline after thorough disinfection andwithout injuring the muscle. The peritoneal cavity was lavaged withsterile saline solution (100 mL) and analyzed for in vivo NET formation,bacteriology, and leukocyte accumulation. NETs in the peritoneal fluidwere qualitatively and quantitatively analyzed using live cell imagingwith confocal microscopy and histone release assays. NETs on the innersurface of the peritoneal membrane were assessed quantitatively usinglive cell imaging followed by standardized grid analysis of 5 randomlyselected high power visual fields per tissue sample (IMAGE-J software,NIH). Peritoneal bacterial colony forming units (cfu) counts werequantified by permeabilizing all recovered leukocytes with 0.1% Triton-X100 for 10 minutes and performing serial dilutions and bacterialcultures on 5% sheep blood agar plates (HARDY DIAGNOSTICS). After a24-hour incubation, bacterial counts were determined. Total leukocytecounts in the peritoneal lavage were determined in Neubauer chambersusing an optical microscope after dilution in Turk's solution (2% aceticacid). Differential leukocyte analysis was performed using a 60× oilimmersion objective to assess morphology of cyto-centrifuged cellsstained with May-Grunwald-Giemsa dye.

Example 23—Assaying Chemotaxis

Potential CRISPP-mediated effects on key neutrophil activities besidesNET formation were assessed: chemotaxis, phagocytosis, reactive oxygenspecies generation, and bacterial killing. It was found that nNIF andCRISPP did not significantly inhibit PMN chemotaxis, phagocytosis, orreactive oxygen species generation (see FIGS. 18A-18C). Total,phagocytotic, and NET-mediated bacterial killing of a pathogenic strainof E. coli was also assessed. It was found that while CRISPPpre-incubation of LPS-stimulated PMNs significantly decreased total andNET-mediated extracellular bacterial killing in comparison to controlPMNs, the phagocytotic component of bacterial killing was not differentbetween the three groups (see FIG. 18D).

Chemotaxis by PMNs isolated from healthy adult donors was assessed usinga modified Boyden Chamber assay±a one hour pre-incubation with nNIF (1nM), CRISPP (1 nM), or SCR (1 nM). Recombinant human IL-8 (2 ng/mL) wasused as the chemoattractant. Chemotaxis through a 5 micron filter wasdetermined by counting PMNs in 10 randomly selected high-power fields aspreviously described in Hill et al., Lancet, 1974, 2: 617-619. Inseparate experiments, nNIF, CRISPP, or SCR (all at 1 nM) were evaluatedfor chemoattractant activity using the same system.

Example 24—Assaying Phagocytosis

As discussed above, it was found that nNIF and CRISPP did notsignificantly inhibit phagocytosis activity in neutrophils. PMNs wereisolated from blood of healthy adult donors and resuspended in M-199 ata concentration of 2×10⁶ cells/mL. Leukocytes were pre-incubated for 60minutes under standard conditions with cytochalasin B and D (10 μM),nNIF (1 nM), CRISPP (1 nM), or SCR (1 nM). Following pre-incubation,PMNs were incubated with 6×10⁶ E. coli BIOPARTICLES (E-13231, MOLECULARPROBES) on a rotator for 4 hours under standard conditions. The PMNswere then washed and resuspended in the starting volume in M-199 beforebeing spun down onto glass coverslips and fixed with 2% paraformaldehydefor 10 minutes and permeabilized with 0.1% Triton-X-100 for 10 minutes.Leukocytes were stained with WGA 555 (INVITROGEN) and TOPRO-3 (MOLECULARPROBES) and randomly selected high power visual field images werecaptured using confocal microscopy. IMAGE-J software (NIH) was used todetermine the percentage of PMNs that were positive for fluorescentlylabeled E. coli BIOPARTICLES detected at 488 nm.

Example 25—Assaying Reactive Oxygen Species Generation

Also, as discussed above, it was found that nNIF and CRISPP did notsignificantly inhibit ROS generation activity in neutrophils. Human PMNsisolated from healthy adult whole blood were resuspended to aconcentration of 2×10⁶ cells/mL in M-199 media and pre-incubated±CRISPP(1 nM) or SCR (1 nM) peptide for 1 hour under standard conditions. ThePMNs were then stimulated with LPS (100 ng/mL) for 1 hour, washed, andresuspended with a dihydrorhodamine (7.25 mM; D-632, MOLECULAR PROBES)and catalase (1000 Units/mL; C-40, SIGMA) mixture and incubated at 37°C. for 10 minutes. After incubation, samples were placed at 4° C. andanalyzed for ROS-dependent fluorescence using a BECTON-DICKINSON FACSCANdevice equipped with CELLQUEST software.

Example 26—Assaying Platelet Activation

Platelets reportedly play a role in regulating NET formation by humanPMNs (see Clark et al., Nat Med, 2007, 13: 463-469). It was determinedwhether CRISPP alters platelet activation and platelet-PMN interactions.P-selectin expression was determined by platelets pre-incubated withCRISPP and SCR peptides followed by stimulation with thrombin. It wasfound that CRISPP and SCR peptides did not alter platelet p-selectinexpression when given alone or following thrombin stimulation (see FIG.18E). Furthermore, in experiments where LPS-stimulated PMNs were mixedwith thrombin-stimulated platelets isolated from the same donor, CRISPPand SCR did not alter platelet/PMN association as assessed via flowcytometry (see FIG. 18F). To further assess whether platelets take upCRISPP from the inflammatory milieu, CRISPP and SCR peptides weresynthesized with a FLAG tag motif added to the carboxy terminus of eachpeptide (CRISPP-F, SCR-F). It was then demonstrated that the CRISPP-Fmaintains NET-inhibitory activity similar to that of un-tagged CRISPP(see FIGS. 21A and 21B), and immunocytochemical experiments wereperformed with freshly isolated human platelets to determine whetherplatelets take up CRISPP-F from the inflammatory milieu. It was foundthat platelets do not take CRISPP-F or SCR-F into their cytoplasm (seeFIG. 18G) while PMNs co-incubated with platelets do. This may suggestthat the NET inhibitory effects of nNIF and CRISPP do not result fromalterations in platelet activation or signaling to trigger NET formationby PMNs.

Using protocols modified from van Velzen, et al., Thromb Res, 2012, 130:92-98, human platelets (1×10⁸ cells/mL) were incubated with CRISPP orSCR (0.1-10 ng/ml) for 1 hour followed by stimulation with thrombin (0.1IU/mL) or control M-199 media for 15 minutes. The cells were preparedfor FACS analysis with the following primary, monoclonal antibodies for20 minutes: PE-conjugated CD41a (platelet-specific marker) andFITC-conjugated CD62P (P-selectin). Isotype and fluorochrome matchedcontrol antibodies were used. After incubation at room temperature inthe dark, the cells were lysed with FACS lysis buffer and analyzed byflow cytometry (BECTON-DICKINSON FACSCAN, CELLQUEST software).

Example 27—Assaying Platelet/PMN Aggregation

Using protocols modified from Saboor et al., Malays J Med Sci, 2013, 20:62-66, human PMNs (2×10⁶ cells/mL) were isolated and incubated withCRISPP or SCR (0.1-10 ng/ml) for 1 hour prior to addition of freshlyisolated human platelet at a ratio of 100:1 for another 15 minuteincubation. After this period, the cells (PMNs and platelets) werestimulated with thrombin (0.1 IU/mL) or control M-199 media for 1 hour.The cells were stained for 20 minutes with the following primarymonoclonal antibodies: PE-conjugated CD41 and FITC-conjugated CD16.Isotype and fluorochrome matched control antibodies were used. Flowcytometry was performed using BECTON-DICKINSON FACSCAN and CELLQUESTsoftware.

Example 28—Assaying Nuclear Decondensation

A possible mechanism by which nNIF and CRISPP inhibit NET formation wasascertained. Using techniques modified from Papayannopoulos et al., JCell Biol, 2010, 191: 677-691, nuclear area assays were used forPMA-stimulated PMNs as a surrogate for NET formation. PMNs werestimulated with PMA (20 nM) for 2 hours and then visualized via livecell imaging with a cell permeable DNA dye. Images from 5 randomlyselected high power fields were captured and the nuclear area of allPMNs on each visual field was quantitated using IMAGEJ software. Nuclearareas were compared for PMNs stimulated with PMA with PMNs pre-incubatedwith CRISPP (1 nM), SCR (1 nM), or the NE inhibitor Sivelestat (200 nM).Qualitative and quantitative results of these assays demonstrated asignificant increase in nuclear area for PMA-stimulated PMNs as comparedto control PMNs or PMNs pre-incubated with each of the three reagentsbut without PMA (see FIG. 19A). Pre-incubation of PMA-stimulated PMNswith CRISPP and Sivelestat, however, resulted in a significant decreasein nuclear area compared to PMA alone while no change was seen in theSCR group (see FIGS. 19A and 19B). Next, immunocytochemical experimentsdesigned to see whether CRISPP is taken up by human PMNs were performed.The CRISPP-F and SCR-F peptides and FLAG-tag specific primary antibodieswere used to assess cellular location. It was found that CRISPP-F andSCR-F are taken up into the cytoplasm by PMNs during a one hourincubation (see FIGS. 18G and 19C). No evidence was found of nucleartranslocation of CRISPP-F or SCR-F (see FIGS. 18G, 19C, and 19D). Theseresults may suggest that NRPs rapidly enter PMNs but not platelets (seeFIGS. 18G, 19C, and 19D), and that uptake of NRPs by PMNs is a functionof size and may not be dependent on amino acid sequence or specificreceptor-mediated transport. Furthermore, protein co-localization assaysusing the DUOLINK protein proximity assay (see Carlo et al., FASEB J,2013, 27: 2733-2741) and primary antibodies targeting F-CRISPP and NE,demonstrate that F-CRISPP and NE can co-localize within 40 nm of eachother following CRISPP incubation for 1 hour (see FIG. 19D). CRISPP caninhibit nuclear chromatin decondensation, which is consistent with theselective inhibition of NET formation by CRISPP and nNIF. This processmay involve inhibition of NE activity.

PMNs were isolated and resuspended to 2×10⁶ cells/mL in M-199 media.They were pre-incubated with CRISPP (1 nM), SCR (1 nM), or theneutrophil elastase inhibitor Sivelestat (100 μM) under standardconditions. Under the same conditions, cells were treated±PMA (20 nM) onpoly-L-Lysine coated glass coverslips for 2 hours. 5 randomly selectedhigh power visual fields per sample were captured via confocalmicroscopy and analyzed for nuclear area using the cell-permeable,fluorescent DNA dye SYTO Green. The nuclear pixel areas of >100individual cells per high power field were determined using IMAGE-Jsoftware (NIH).

Example 29—Determining CRISPP Peptide Cellular Localization

The cellular locations of FLAG-tagged CRISPP (F-CRISPP) and FLAG-taggedSCR (F-SCR) peptides were determined using immunocytochemistry. Adultneutrophils were pre-incubated with either F-CRISPP (1 nM) or F-SCR (1nM) for 1 hour under standard conditions followed by stimulation withLPS (100 ng/mL) for 2 hours. The PMNs were then spun down onto glasscoverslips with 2% p-FA fixation and 0.1% Triton-X-100 permeabilization.FLAG-tagged peptide was detected using a monoclonal anti-FLAG antibody(F1804, SIGMA) with TOPRO-3 as a nuclear counterstain.

Example 30—Using a DUOLINK Protein Proximity Assay

The DUOLINK Protein Proximity Assay (SIGMA) was used to determinewhether CRISPP and NE co-localize within PMNs isolated from healthyadults. PMNs were isolated and pre-incubated for 1 hour with F-CRISPP(10 nM) or F-SCR (10 nM) under standard conditions prior to stimulationwith LPS (100 ng/mL) on poly-L-lysine coated glass coverslips. TheDUOLINK Protein Proximity Assay was performed using a rabbitanti-neutrophil elastase antibody (ab131260, ABCAM) and a mouseanti-FLAG antibody (F1804, SIGMA). Fluorescence detected via confocalmicroscopy at 546 nm indicated FLAG-tagged peptide and NEco-localization.

Example 31—Assaying Bacterial Killing

As discussed above, it was found that while CRISPP pre-incubation ofLPS-stimulated PMNs significantly decreased total and NET-mediatedextracellular bacterial killing in comparison to control PMNs, thephagocytotic component of bacterial killing was not different betweentotal, phagocytotic, and NET-mediated bacterial killing of a pathogenicstrain of E. coli (see FIG. 18D).

Bacterial killing efficiency of primary human PMNs was determined aspreviously described in Yost et al., Blood, 2009, 113: 6419-6427. PMNswere incubated for 30 minutes at 37° C. in 5% CO₂/95% air alone or withthe phagocytosis inhibitors cytochalasin B and D (10 μM). The leukocyteswere then stimulated with LPS (100 ng/mL), placed inpoly-L-lysine-coated wells of a 24-well tissue culture plate, andincubated at 37° C. for 1 hour to induce cellular activation andformation of NETs. To inhibit NET-mediated bacterial killing, weincubated selected wells with DNase (40 U/mL) for 15 minutes prior toaddition of bacteria. E. coli (1 cfu/100 PMN) were added to the PMNs,followed by continued incubation for 2 hours. The PMNs were thenpermeabilized with 0.1% Triton-X 100 for 10 minutes and each well wasscraped to free all cells. Serial dilutions were performed and bacterialcultures were grown on 5% sheep blood agar plates (HARDY DIAGNOSTICS).After a 24-hour incubation, bacterial counts were determined. Total,phagocytotic, and fractional NET-mediated bacterial killing weredetermined as described above.

Example 32—Studying Survival in Murine Models of Sepsis

Animals were treated with CRISPP (10 mg/kg), nNIF (10 mg/kg), or SCR (10mg/kg) by i.p. injection 1 hour prior to and 6 hours after infection (E.coli, 4.5×10⁷ cfu/mouse, i.p. injection) or stimulation (LPS, 25 mg/kg,i.p. injection). Other mice were subjected to CL/P as a model ofpolymicrobial sepsis (see Araujo et al., Microvasc Res, 2012, 84:218-221). Still other mice were injected with nNIF, CRISPP, or SCR 4hours after injection/stimulation to evaluate post-infection impact ofnNIF/CRISPP on survival. Control mice were injected with saline aloneand control mice for the CL/P mice were subjected to sham surgery. Themice in these studies were not treated with fluid resuscitation orantibiotic treatment, but were provided with ample food and water duringthe experiments. Survival was assessed over 6 days at 6 hour to 12 hourintervals.

Example 33—Performing Statistical Analysis

GRAPHPAD PRISM statistical software (version 4, GRAPHPAD SOFTWARE) wasused to analyze results. Reported values are the mean±SEM. To assessdifferences between more than two sets of normally distributed data theone-way ANOVA test with Tukey's or Bonferroni's post-hoc testing wasemployed. For comparison of two groups of continuously distributed data,the unpaired, single or two tailed Student's t-test was used whereappropriate. For comparison of murine survival curves, the Log-rank(Mantel-Cox) test was used. All P values of less than 0.05 wereconsidered statistically significant.

It will be apparent to those having skill in the art that many changesmay be made to the details of the above-described embodiments withoutdeparting from the underlying principles of the invention. The scope ofthe present invention should, therefore, be determined only by thefollowing claims.

The invention claimed is:
 1. A method for treating neutrophilextracellular trap (NET) mediated inflammatory tissue damage in apatient, comprising: administering to the patient an effective amount ofa pharmaceutical composition comprising a peptide consisting of theamino acid sequence of SEQ ID NO:4 having an amino acid substitution anda pharmaceutically acceptable carrier to reduce a pathological effect orsymptom of the NET-mediated inflammatory disorder.
 2. The method ofclaim 1, wherein the pharmaceutical composition substantially inhibitsNET-mediated inflammatory tissue damage.
 3. The method of claim 1,wherein the patient is human.
 4. A method for inhibiting NET formationin a patient, comprising: administering to the patient an effectiveamount of a pharmaceutical composition comprising a peptide consistingof the amino acid sequence of SEQ ID NO:4 having an amino acidsubstitution and a pharmaceutically acceptable carrier to substantiallyinhibit NET formation.
 5. The method of claim 4, wherein the NETformation is stimulated by a virus selected from at least one of ahemorrhagic fever virus, a Filovirus, an arenavirus, a hantavirus, aflavivirus, dengue virus, yellow fever virus, or HIV-1.
 6. The method ofclaim 4, wherein the NET formation is stimulated by a bacterium selectedfrom at least one of a Bacillus species, an Escherichia species, aFrancisella species, a Staphylococcus species, a Streptococcus species,or a Yersinia species.
 7. The method of claim 4, wherein the patient ishuman.
 8. A pharmaceutical composition, comprising: a peptide consistingof the amino acid sequence of SEQ ID NO:4 having an amino acidsubstitution; and a pharmaceutically acceptable carrier.
 9. The methodof claim 1, wherein at least one amino acid of the peptide is bound to achemical modifier, and wherein the chemical modifier is selected from atleast one of a lipid, a polyethylene glycol (PEG), or a saccharide. 10.The method of claim 1, wherein the peptide does not globally depresspolymorphonuclear leukocyte (PMN) function.
 11. The method of claim 1,wherein the peptide does not substantially inhibit one or moreactivities of a polymorphonuclear leukocyte (PMN) selected from thegroup consisting of chemotaxis, chemokine synthesis and secretion,cytokine synthesis and secretion, extracellular bacterial killing,intracellular bacterial killing, phagocytosis, and reactive oxygenspecies (ROS) generation.
 12. The method of claim 1, wherein the peptideis present in an amount effective to substantially inhibit neutrophilextracellular trap (NET) formation.
 13. The method of claim 1, whereinthe NET formation is stimulated by at least one of a bacterium, afungus, a parasite, or a virus.
 14. The method of claim 1, wherein theamino acid substitution is a nonconservative substitution.
 15. Themethod of claim 4, wherein the amino acid substitution is anonconservative substitution.